Final Report

Project title (Acronym) Assessment of Dickeya and Pectobacterium spp. on vegetables and ornamentals (Soft rot)

Project duration:

Start date: 2015-01-01

End date: 2018-12-31

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Contents

1. Research consortium partners ...... 3 2. Short project report...... 6 2.1. Executive Summary ...... 6 2.2. Project aims ...... 7 2.3. Description of the main activities and results ...... 8 2.3.1. Meetings ...... 8 2.3.2. Diagnostic methods ...... 8 2.3.3. Surveys and risk assessments ...... 8 2.3.4. Plant pathogen interactions ...... 11 2.3.5. Disease management ...... 11 2.4. Conclusions and recommendations to policy makers ...... 13 2.5. Benefits from trans-national cooperation ...... 13 3. Publications ...... 15 3.1. Article(s) for publication in the EPPO Bulletin ...... 15 3.2. Article for publication in the EPPO Reporting Service ...... 15 3.3. Article(s) for publication in other journals ...... 15 4. Open Euphresco data ...... 17

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1. Research consortium partners

Coordinator – Partner 1 Organisation Wageningen University & Research (WUR)

Name of Contact Jan van der Wolf Gender: M (incl. Title) Job Title Bacteriologist, Senior Scientist Postal Address P.O. Box 16, 6700 AA Wageningen, the Netherlands E-mail [email protected] Phone +316 10028194

Partner 2 National Plant Protection Organization, Netherlands Food and Consumer Products Organisation Safety Authority (NVWA) Name of contact Dr Maria Bergsma-Vlami Gender F (incl. Title) Job Title Bacteriologist, head of the bacteriology team (Postal) address Geertjesweg 15, Wageningen, The Netherlands E-mail [email protected] Phone +316 15891907

Partner 3 Organisation Science and Advice for Scottish Agriculture (SASA) Name of contact Prof. dr Gerry Saddler Gender: M (incl. Title) Job Title Bacteriologist, Director SASA Postal address SASA, Roddinglaw Road, Edinburgh, Scotland, UK E-mail [email protected] Phone +44 (0)1312448925

Partner 4 Organisation French Association of seed potato producers (FN3PT-RD3PT) Name of contact Dr Valérie Hélias Gender: F (incl. Title) Job Title Phytopathologist, Researcher, Group leader UMR 1349 IGEPP INRA - Agrocampus Ouest Rennes - Université Rennes 1 Postal address BP 35327 F-35653 LE RHEU FRANCE E-mail [email protected] Phone +33 (0)223485186

Partner 5 Organisation ARO - Volcani Center (ARO)

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Name of contact Dr. Leah Tsror; Dr Iris Yedida Gender: F; F (incl. Title) Job Title Senior Scientist, Potato Disease expert; Bacteriologist, Senior Scientist Department of Plant Diseases and Weed Research, ARO (Volcani), Gilat Research Center, M.P. Negev, 85250, Israel; The Institute of Plant Sciences, Postal address Ornamental Plants and Agricultural Biotechnology Dep. ARO, The Volcani Center, POB 15159 Hamakabim Road 68, 7528809 Rishon Lezion, Israel E-mail [email protected] ; [email protected] Phone +972 (0)89928660; +97239683387

Partner 6 Organisation University of Helsinki (UH) Name of contact Dr Minna Pirhonen Gender: F (incl. Title) Job Title Bacteriologist, University lecturer Department of Agricultural Sciences, PO Box 27, 00014 University of Helsinki, Postal address Finland E-mail [email protected] Phone +358 504922077

Partner 7 Organisation Natural Resources Institute Finland (LUKE) Name of contact Dr. Yeshitila Degefu Gender: M (incl. Title) Job Title Bacteriologist, Senior Scientist Postal address Paavo Havaksen tie 3, 90014 University of Oulu E-mail [email protected] Phone +358 295326068

Partner 8 Organisation Potato Research Institute (PETLA) Name of contact Jussi Tuomisto Gender: M (incl. Title) Job Title Director PETLA Postal address Potato Research Institute, Alapääntie 104, 61400 Ylistaro, Finland E-mail [email protected] Phone +358 404507200

Partner 9 Intercollegiate Faculty of Biotechnology University of Gdansk & Medical University Organisation of Gdansk (UG) Job Title Bacteriologist, University Professor

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Name of contact Prof. Dr Ewa Lojkowska Gender: F (incl. Title) Postal address Abrahama str. 58, 80-307 Gdansk, Poland E-mail [email protected] Phone + 48 725991070

Partner 10 Organisation Canadian Food Inspection Agency (CFIA) Name of contact Dr Sean Li Gender: M (incl. Title) Job Title Bacteriologist, Senior Scientist Postal address 93 Mount Edward Road, Charlottetown, PE, Canada, C1A5T1 E-mail [email protected] Phone +1 9023680950 ext. 263

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2. Short project report

2.1. Executive Summary Dickeya and Pectobacterium belonging to the group of soft rot Pectobacteriaceae (SRP) are causing emerging problems in a wide range of vegetable and ornamental crops in Europe, including potato, carrot, cabbage, Chinese cabbage, celery, leek, pepper, parsley, Zantedeschia, hyacinth, Dahlia, Chrysanthemum, Philodendron, Freesia, Saintpaulia, Iris, Aglaonema, Crocus, Campanula and Phalaenopsis. The phytopathogens in both genera are genetically and phenotypically highly diverse. Disease problems in the different hosts are associated with the introduction of new variants or by spread of groups already present in Europe. Within this Euphresco project we aimed to identify and assess the risks of these new variants, and to develop management strategies, including reliable diagnostic methods to prevent introductions and further spread of SRP. To reach our goals, meetings were organized and collaborations were established with specialists worldwide. All information on meetings, protocols and activities of the Euphresco group are published on the Dickeya/Pectobacterium website, conveyed by the James Hutton Institute in Invergowrie (Scotland) (https://engage.hutton.ac.uk, contact person Dr I. Toth/Dr J. Fairly).

During the project, 1.5 days meetings were held in 2015 in Gdansk (Poland), in 2016 in Helsinki (Finland), in 2017 in Edinburgh (Scotland) and in 2018 in Emmeloord (The Netherlands). Meetings were attended by an average of 30 participants from organizations in EU member states, North- and Latin America, Africa, Asia and Australia.

One project’s objective was to develop methods for the detection and identification of Pectobacterium and Dickeya species in different matrices. For this, a panel of reference strains has been compiled for Dickeya and Pectobacterium species. Most strains have been deposited in international collections. For most strains also whole genome sequence data are available. During the course of this project, several diagnostic tests were developed and evaluated, often based on the TaqMan technology.

In several countries, surveys in potato and ornamental crops were conducted, but also in other matrices of the potato ecosystem, including water used for irrigation. In addition, new taxonomic groups that have been identified, are now new species including: P. versatile, P. aquaticum, P. fontis and P. polonicum. In potato, P. brasiliense became dominant as blackleg causing organism and has largely outcompeted D. solani in the last five years. In surface water in Europe, D. zeae was found to be the dominant SRP. In other continents, serious outbreaks of potato blackleg with other SRP has been reported, such as D. dianthicola in the USA and Australia. Various new SRP have been described, namely P. punjabense, P. peruviense, P. polaris, D. lacustris and D. zantedeschia. For the first time, D. fangzhongdai was described in Phalaenopsis. Not all species can cause potato blackleg.

Studies on various virulence factors were conducted for SRP, such as on chemoreceptors, small phenolic plant compounds interacting with signal molecules of Pectobacterium involved in the quorum sensing mechanism of the pathogen. A Tn-seq approach was developed and used to identify new virulence factors.

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Information was exchanged on disease management strategies which include cultivation practices, resistance breeding, hygiene and the use of (bio-) control agents. A strict hygiene and an intensive monitoring of seed lots was found to be associated with a significant reduction of infections with blackleg causing SRP. A phage therapy has been developed to protect (seed) potato tubers against soft rot during storage. Various bacteriophages and bacterial antagonists were characterized and some evaluated for control of potato soft rot and/or blackleg. Steam treatments for seed tubers were found to decrease the blackleg incidence. Cold plasma treatment was found to kill SRP grown in vitro. Similarly, stabilized silver nanostructures killed SRP. It was found that seed potato lots can differ in suppressiveness against D. solani. Indications were found that the microbiome in tuber tissue plays a role in this.

2.2. Project aims Given the range of current or emerging Dickeya and Pectobacterium problems, there was a need to optimise resources by ensuring that research is well coordinated and is not duplicated nationally and internationally. The consortium searched for opportunities to cluster existing work to ensure added value through joined meetings, appropriate joint outputs and inclusion of appropriate additional work.

The overall goal of the project 2015-D-135 ‘Assessment of Dickeya and Pectobacterium spp. on vegetables and ornamentals (Soft rot)’ was to identify and assess the risks of new variants of soft rot Pectobacteriaceae and new diseases caused by SRP, and to develop management strategies, including reliable diagnostic methods to prevent introductions and the further spread of SRP.

The project had the following objectives:

Diagnostic methods. To develop and identify reliable and cost-effective methods for the detection and identification of Pectobacterium and Dickeya species in different matrices. Development of protocols has partly been achieved within previously funded Euphresco projects on Dickeya and Pectobacterium1.

Risk assessment. To assess the presence of different Dickeya and Pectobacterium species in vegetable and ornamental crops and in environmental/industry sources such as machinery, irrigation water as part of the risk assessment. For these surveys, a representative panel of SRP strains were compiled. Collected strains were characterised using molecular and phenotypic methods. Risk assessment also involved generating new information on the ecology of SRP in different hosts, including knowledge on dissemination and infection pathways, aggressiveness, survival strategies and plant pathogen interactions.

Disease management. To increase knowledge on disease management strategies via exchange of information, which includes information on cultivation practices, resistance breeding, hygiene and the use of (bio-) control agents.

1 Dickeya species in potato and management strategies https://zenodo.org/record/1326445#.Xg8MnUdKjct Assessment of Dickeya and Pectobacterium spp. on potatoes and ornamentals https://zenodo.org/record/1326504#.Xg8Mx0dKjct

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2.3. Description of the main activities and results The references refer to the abstracts published during the various meetings, see Annex I, II, III, IV

2.3.1. Meetings The Euphresco consortium on SRP had been successfully collaborating since 2013. During the Euphresco project 2015-D-135, 1.5 days meetings were held in 2015 in Gdansk, in 2016 in Helsinki, in 2017 in Edinburgh and in 2018 in Emmeloord. Meetings were attended by an average 30 participants from EU member states, North- and Latin America, Africa, Asia and Australia. The exchange of information between scientists, members from national plant protection organizations, breeders and policy makers was of great support in finding novel strategies for disease management. The consortium agreed to continue as an informal group, which will be coordinated by Prof. Dr. R. Czajkowski (UGdansk). From 2019 onward, no new Euphresco project on SRP will be conducted.

2.3.2. Diagnostic methods One goal was to develop and fine-tune reliable and cost-effective methods for the detection and identification of Pectobacterium and Dickeya species in different matrices. For this, a panel of reference strains was compiled for Dickeya and Pectobacterium species. The majority of the strains were deposited in international collections. For most strains also whole genome sequence data are available. The various diagnostic methods were extensively discussed including 1. isolation, 2. characterization of bacteria using multi-locus sequence analysis, 3. testing for virulence, 4. enrichment of SRP, 5. detection with TaqMan, LAMP tests and microarray techniques, 6. multiplex detection with the (Luminex) xTaq technology, and 7. High- Throughput Sequencing (HTS)-analysis in complex substrates. During the course of this project, several new diagnostic tests were developed and evaluated, often based on the TaqMan technology. For example, in the Netherlands new TaqMan tests were developed for P. brasiliense and P. parmentieri (Van der Wolf, 2015). In France, 11 real-time PCR tests were evaluated in comparative testing, including tests for P. atrosepticum, Dickeya spp., D. solani, D. dianthicola, P. brasiliense and P. parmentieri (Hélias, 2016). Pectinolytic bacteria isolated from blackleg diseased plants can be easily and rapidly identified by gapA sequencing (Cigna et al., 2017). D. solani can be easily identified using potato dextrose agar on which the pathogen grows as wrinkled and brownish colonies (Pirhonen, 2017). It was found that post- harvest seed lot testing can be reliably used to predict stand losses due to infections with D. dianthicola in the next growing season (Secor, 2016).

Unfortunately, a Test Performance Study (TPS) for detection of SRP in potato, to be organized by the Dutch National Plant Protection Organization (NVWA), had to be cancelled.

2.3.3. Surveys and risk assessments Surveys in potato and ornamental crops were conducted in several countries, and the presence of SRP was also investigated in the potato ecosystem, including sources of irrigation water, using the diagnostic methods described previously. In Poland, P. parmentieri was dominant in diseased plants, but also infections with P. atrosepticum, P. carotovorum, D. solani and D. dianthicola were found (Lebecka et al., 2015). In contrast, in surface water sampled in Poland, only D. zeae and D. chrysanthemi were found (Potrykus et al., 2015). In Norway, P.

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carotovorum and P. atrosepticum were dominant in diseased plants whereas only in a single case infection with P. parmentieri and D. solani were found (Lebecka et al., 2015). In Switzerland, P. brasiliense was the most prevalent in blackleg symptomatic plants, followed by P. carotovorum, P. parmentieri, D. dianthicola and D. solani (Dupuis et al., 2017). In Finland a group of strains was isolated from potato samples which were similar to, but nevertheless differentiable from P. polaris and may represent a new species or subspecies of P. polaris (Pirhonen et al., 2018). In the Netherlands, P. brasiliense is dominant in blackleg diseased plants and symptomless infected seed tubers (Vreeburg, 2017). Strains isolated from diseased plants were mainly virulent, but non-virulent and hypovirulent strains were isolated from latently infected tubers. P. brasiliense was also detected in and isolated from insects. In comparative testings in Switzerland, D. solani was the most aggressive one, whereas strains of P. carotovorum did not induce blackleg symptoms. In Scotland, still P. atrosepticum is the main causative organism of potato blackleg, although incidentally P. parmentieri and P. brasiliense are detected (Saddler, 2015, Cahill, 2016). Infections occurred in Scotland already in the first field generation. In Israel, diseased plants were infected with D. solani and P. brasiliense (Tsror, 2015). In France, beside several Pectobacterium spp., D. solani and D. dianthicola were found in potato (Pedron et al., 2015; Hélias et al., 2016). The group of D. solani strains could be subdivided in two groups on the basis of single nucleotide polymorphism SNP’s, indels and additive horizontal gene transfers. Several newly identified groups, that are now species, as P. versatile (Hélias et al., 2018) were found in culture collections; P. punjabense isolates were sporadically identified from French and Dutch laboratory collections (Cigna et al., 2018). A P. punjabense strain from Pakistan was able to cause blackleg. In Chile, P. atrosepticum, but likely also other SRP variants (P. carotovorum and D. dadantii) are responsible for high losses of up to 30% in potato production (Acuna et al., 2015, 2016, 2017). A soft rot causing strain isolated from potato in Peru was found to be a new species and was named P. peruviense. The same species was also isolated from alpine rivers in France. In the USA, blackleg is caused predominantly by D. dianthicola, which seem to originate from different sources as the isolates were diverse (Hao et al., 2016, 2018; Secor et al., 2017). In the USA, Dickeya dianthicola was found in potatoes in the stolon end, the sprout, but most frequently in the peel strip, indicating that testing of the stolon end is not sufficient (Secor et al., 2017). In Canada, D. dianthicola, P. parmentieri, P. atrosepticum and P. carotovorum are the most prevalent blackleg causing agents (Li, 2016). In South Africa, water samples from dams, boreholes, rivers and overhead irrigation systems in five potato growing areas were surveyed for Pectobacterium and Dickeya species. Identification was done by REP-PCR and Multiplex PCRs, using species specific primers. Initial tests revealed that several bacteria are detectable in water systems used for irrigation in most regions sampled, among which P. brasiliense, P. atrosepticum, P. carotovorum and Dickeya spp. (Laughton & Van der Waals, 2016; Van der Waals et al., 2016). A high variation of SRP are found in water ways in France, including a new Dickeya species named D. lacustris (Bertrand, 2017; Pedron et al., 2018; Hugouvieux-Cotte-Pattat et al., 2018). Introduction of virulent variants present in water ways may pose a threat to agriculture and horticulture. In 2017 in Australia, an outbreak of Dickeya dianthicola in (seed) potatoes was reported. Likely, infected Dahlia tubers were the initial infection source. The pathogen was also detected in freesia bulbs (Mann et al., 2017).

In Phalaenopsis grown in Slovenia, Dickeya sp. were isolated which were similar to D. fangzhongdai found in China, that causes a bleeding canker of pear (Alic et al., 2016). A soft 9 Euphresco project report

rot causing strain isolated from Calla lilly was found to be a new species and was named P. zantedeschiae (Waleron et al., 2018).

An important issue was the sudden changes in the population structure of SRP in potato in Europe. Between 2000 and 2010, D. solani was the most dominant causative agent of potato blackleg in the seed production in several countries on the European continent, but in the last 5 years P. brasiliense became increasingly prevalent in Switzerland and elsewhere (De Werra, 2015). Nevertheless, outbreaks as a result of infections with D. solani are still reported (Degefu, 2015). Research was conducted to find the driving force behind it. P. brasiliense may originate from alternative host plants, including weeds. In Israel, the pathogen was isolated from latently infected Malva nicaeensis plants (Tsror et al., 2019).

The population of D. solani strains in Europe is still highly clonal, although the intraspecific diversity is slowly growing (Pedron et al., 2017; Lojkowska et al., 2017; Van Gijsegem, 2018). The global population of P. atrosepticum strains, although phenotypically similar, showed a high intraspecific variation (Pritchard and Toth, 2017). Similarly, a high genetic variation was found between strains of P. parmentieri (Lojkowska et al., 2017, 2018).

The survival of P. brasiliense in soil was studied in pot trials under different temperature and moisture regimes (Van der Waals et al., 2015). Six weeks after inoculation, no culturable cells could be detected, although after 5 months positive reactions were still found in a real-time PCR test. In Scotland, it was shown that P. atrosepticum (a related pathogen) was able to move in a seed production field during the growing season from diseased to healthy plants (Campbell et al., 2015). Free-living nematodes were found able to disperse soft rot bacteria which may play a role in the observed spread of the pathogen in soil (Pirhonen et al., 2015).

During flailing (mechanical haulm destruction) of a SRP infected seed potato crop, contaminated aerosols could be easily trapped at a short distance from the crop with an air samplers, indicating aerosols as a potential (initial) infection source. Aerosols can be wind blown over long distances (up to 1 km) and potentially infect adjacent crops (Van der Wolf et al., 2018).

A natural solution to provide safe irrigation water is managed aquifer recharge (MAR) for agriculture. Here, tile drainage water (TDW) is collected and infiltrated in brackish aquifers, resulting in a fresh water ‘bubble’ in the subsurface which gives farmers access to sufficient fresh water to irrigate their crops even in times of drought. To prevent the occurrence of crop diseases, the removal of selected plant pathogens in TDW and during aquifer soil passage is required. Under aerobic conditions D. solani and P. carotovorum were not detected anymore in 0.1-ml samples within 14 days at 10 ⁰C. D. solani and P. carotovorum were no longer detected within 6 days at 25 ⁰C (Eisfeld et al., 2018)

Field experiments in the Netherlands using vacuum infiltrated tubers showed that P. atrosepticum and some strains of P. brasiliense were highly aggressive, D. solani and P. parmentieri were moderately aggressive, whereas four different P. carotovorum strains did not induce blackleg symptoms (Van der Wolf et al., 2015). This lack of virulence for P. carotovorum was also found in field experiments in Finland (Pasanen & Pirhonen, 2015)

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2.3.4. Plant pathogen interactions Within the frame of risk assessment, studies on molecular plant pathogen interactions were also conducted. Chemoreceptors were identified and the response to various compounds were studied (Hugovieux Pattat, 2016). Small phenolic plant compounds in Pectobacterium were found to influence biofilm formation via the quorum sensing mechanism (Joshi et al., 2015; Yedidia et al., 2016, 2017). External application of the signal molecules resulted in a full recovery of the infection capacity. In P. brasiliense, compounds were found that supported the pathogen to compete with other bacteria in planta (Moleleki, 2016). P. atrosepticum was localized as emboli in xylem vessels, multicellular structures of potato plant and bacterial compounds, to protect the bacteria from recognition by the defence mechanism of the host (Gorshkov et al., 2017). The production of coronafacic acid is important in the transition from the ‘amenable’ to the ‘devastating’ mode for P. atrosepticum in xylem vessels. For P. atrosepticum also adaptive proliferation was demonstrated that occurs under stress conditions, when the population density is too low to implement cell-to-cell communication. This proliferation is evidently supported by endogenous cell resources (Gorshkov et al., 2018). A transposon sequencing (Tn-seq) approach was developed and used to identify new virulence factors in D. dadantii (Royet et al., 2017).

The expression of virulence determinants of bacterial strains from different hosts is dependent on the tested plant species (i.e. Brassica oleracea or Zantedeschia aethiopica); whether it is a host or a non-host of the strain (Khadka and Yedidia, 2018). Differential gene expression was also demonstrated in the induced-resistance response of the plant hosts to the different bacterial strains.

It was found that repeated cultivation of strains of D. dianthicola, D. solani and P. parmentieri on agar media can result in a loss of virulence. To test for the blackleg causing capacity of strains or the susceptibility of cultivars field studies should be conducted; in vitro maceration assay on potato tubers are not useful for this.

2.3.5. Disease management In the Netherlands, an existing programme of strict hygiene and an intensive monitoring of seed lots resulted in a significant reduction of infections with blackleg causing SRP’s (Kristelijn, 2015).

Broad host range bacteriophages able to infect the dominant Pectobacterium and Dickeya species were isolated after which they were genetically and phenotypically characterized (Czajkowski, 2015, 2016). The potential of bacteriophages was discussed. A phage therapy was developed in Scotland to protect (seed) potato tubers against soft rot during storage (Blackwell, 2017).

In France, 24 potato hybrids originating from three wild resistance donor species (S. andigena, S. brevidens and S. etuberosum) were tested for their susceptibility to soft rot (Hélias et al., 2016). Of these hybrids, 10 showed a very low severity against two out of five SRP species.

Several groups worked on the biocontrol of SRP’s. For a Pseudomonas strain inhibiting SRP’s in vitro, a hypothetical gene coding for an antimicrobial metabolite was knocked out after which the biocontrol strain lost its ability to inhibit the growth of Dickeya spp. in vitro (Krzyżanowska, 11 Euphresco project report

2015). Transcriptome profiling using a Serratia plymuthica biocontrol strain showed that treatment of potato tubers resulted in expression of genes involved in stress responses, hormone signalling and plant defence (Hadizadeh et al., 2016). In France, furrow application with Pseudomonas putida and Rhodococcus erythropolis biocontrol strains resulted in a reduction of the blackleg incidence and a decrease of seed infections (Munier et al., 2017). For the control of soft rot in potato during storage, compatible mixtures of antagonistic bacteria were successfully used (Krzyzanowska et al., 2018).

In Israel, seed tuber treatments were evaluated for their ability to reduce tuber rot during the fall-winter season, especially at early planting during September when temperatures are relatively high. The seed treatments included dry steam, oxolinic acid, mancozeb and MB5K (surface sterilizing agent) applied prior to planting. All treatments reduced seed decay or tuber rot, however, the results were inconsistent in field trials conducted in different years. Direct current atmospheric pressure glow discharge, also known as cold plasma, was shown to eradicate plant pathogenic bacteria, including D. solani (Motyka-Pomagruk et al., 2018). Silver nanostructures stabilised by pectin or sodium dodecyl sulfate (SDS) possessed strong antimicrobial properties against Dickeya and Pectobacterium and may be used in disease management programs (Motyka-Pomagruk et al., 2018).

Seed potato lots can differ in suppressiveness against D. solani. If seed lots from different origin with a low infection rate were vacuum infiltrated with high densities of the pathogen and planted in the same soil, remarkable differences in disease incidence were found (Van der Wolf et al., 2017). Indications were found that the microbiome in tuber tissue plays a role in the suppressiveness. Next studies are focussed on the manipulation of the microbiome.

Within the genus Dickeya, D. solani has been recognized as a threat, particularly for crops grown in hot climates. Furthermore, for many years D. dianthicola has been responsible for losses in various crops, under which potato. D. dadantii has been found in various ornamental crops and occasionally, D. dadantii occurs in potato in Europe. This raises questions about distribution and risks of this Dickeya species. In waterways in the UK and Finland D. aquatica and also D. chrysanthemi and D. zeae in Poland have been found, but the host has not been identified yet. There are also Dickeya variants not present in Europe yet, for which risks have not been assessed, such as D. fangzhongdai found in China, that causes a bleeding canker of pear.

Also, within the genus Pectobacterium (for Europe) new variants were described recently from which some have already a high impact. P. brasiliense and P. parmentieri (earlier wrongly classified as P. carotovorum subsp. carotovorum) are broad host range pathogens that cause emerging problems in potato in Europe. Similarly, P. aroidearum and P. carotovorum subsp. odoriferum, both with a wide host plant spectrum, are recognized to cause economic losses in ornamental plants, particularly in flower bulbs.

Given the range of current or emerging Dickeya and Pectobacterium problems, there is a need to optimise resources by ensuring that research is well coordinated and is not duplicated nationally and internationally. There are opportunities to cluster existing work to ensure added value through joined meetings, appropriate joint outputs and inclusion of appropriate additional work (Dickeya network and Euphresco consortium). 12 Euphresco project report

2.4. Conclusions and recommendations to policy makers This project strongly contributed to a rapid and extensive information exchange on the characterization of (new) soft rot and blackleg causing pathogens, on virulence mechanism, on spread, survival and colonization and on the disease management via hygiene, cultivation measures and via treatments of plants for planting. It further resulted in the availability of improved diagnostic methods for detection and identification. The annual meetings stimulated collaborations of participants in research projects and publication of shared data. The observation that the population structure of soft rot Pectobacteriaceae (SRP) rapidly change, in particular in potato is highly important. One example is the introduction of a subgroup of Pectobacterium brasiliense. This introduction may originate from contaminated insects, rain-water, surface water or from windblown aerosols, but contact infections with (debris of) plants grown in rotation with potato cannot be excluded. The finding and characterization of several new variants of (SRP) indicates that there is risk for introductions of new aggressive soft rot causing pathogens from the environment. It stresses the importance of regular surveys in order to keep diagnostic methods and other management tools methods updated. The high risks for initial infections from environmental sources that can hardly be controlled, including rain-water, indicates the need for management practices that reduce the risk for spread and symptom expression. To reduce spread, the role of hygiene and cultivation methods are of utmost importance, such as reducing the risks for the presence of symptomatic plant material during harvest and post-harvest processes. So far, attempts to select cultivars with a high level of resistance against SRP failed. The use of improved selection and modern breeding techniques may be helpful to develop genotypes with a high, consistent level of resistance. To limit damage, treatments of planting material are highly desirable. A combination of physical treatments to reduce inoculum levels of planting material followed by the application of biological agents (antagonists) able also to control the pathogen inside plants is a strategy that is expected to reduce inoculum load and symptom development. The improved knowledge on molecular plant pathogen interactions, such as the effect of phenolic compounds on the virulence of the pathogen may also result in the selection of cultivars or the development of agents that can reduce damage by SRP.

2.5. Benefits from trans-national cooperation Given the range of current or emerging Dickeya and Pectobacterium problems, there was a need to optimise resources by ensuring that research is well coordinated and is not duplicated nationally and internationally. Via joined annual meetings, this Euphresco initiative strongly supported exchange of information and strains, discussions on disease management and control of soft rot diseases, and collaborations via research projects. Collaborations between members of this group are evidenced by several joined publications published during the course of the project.

The members of the group are highly motivated to meet annually also in the future. In June 2020, they will participate in the 4th International Erwinia Workshop, organized by Prof. Dr. Ian Toth (James Hutton Institute, Scotland) as a satellite meeting of the International Conference on Plant Pathogenic Bacteria in Assisi (Italy). From 2021 onward, the consortium will continue

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to collaborate as an informal Dickeya/Pectobacterium group chaired by Prof. Dr. R. Czajkowski (Gdansk, Poland).

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3. Publications

3.1. Article(s) for publication in the EPPO Bulletin None.

3.2. Article for publication in the EPPO Reporting Service None.

3.3. Article(s) for publication in other journals . Chawki K., Quêtu-Laurent A.G. Taghouti G., Caullireau E., Fischer-Le Saux M., Le Hingrat Y., Andrivon D., Portier P. (2018). The Pectobacterium complex: Diversity and phylogeny. Phytopathology Volume 108, Number 10S : S1.30 . De Boer, S. H., Charkowski, A.O. and Van der Wolf, J.M. (2017). Detection of Pectobacterium spp. and Dickeya spp. in potato tubers. P. 243- 247 In M’Barek Fatmi, Walcott, R.R. and Schaad, N.W. Detection of plant-pathogenic bacteria in seed and other planting material. APS Press, St Paul, USA. . Degefu, Y., Potrykus, M., Golanowska, M., Virtanen, E., and Lojkowska, E. (2013). A new clade of Dickeya spp. plays a major role in potato blackleg outbreaks in North Finland. Annals of Applied Biology 162:231-241 . Degefu, Y., Somervuo, P., Aittamaa, M., Virtanen, E., Valkonen, JPT. (2016). Evaluation of a diagnostic microarray for the detection of major bacterial pathogens of potato from tuber samples. EPPO Bulletin 46:103–111 . Hadizadeha, I., Peivastegana, B., Hannukkala, A., Van der Wolf, J.M., Nissinen, R., and Pirhonen, M. (2019). Biological control of potato soft rot caused by Dickeya solani and the survival of bacterial antagonists under cold storage conditions. Plant Pathology 68, 297– 311 . Hugouvieux-Cotte-Pattat, N., Jacot-des-Combes, C., Briolay, J. (2019). Dickeya lacustris sp. nov., a pectinolytic bacterium isolated from lakes in the French region of La Dombes. Int J Syst Evol Microbiol 69:721-726. . Khayi S., Blin P., Pédron J., Chong T.M., Chan K.G., Moumni M., Hélias V., Van Gijsegem F., Faure D. 2015. Population genomics reveals additive and replacing horizontal gene transfers in the emerging pathogen Dickeya solani. BMC Genomics. 16:788-801. DOI 10.1186/s12864-015-1997-z. . Khayi S., Cigna J., Chong T.M., Quêtu-Laurent A., Chan K.G., Hélias V., Faure D. (2016). Transfer of the potato plant isolates of Pectobacterium wasabiae to Pectobacterium parmentieri sp. nov., International Journal of Systematic and Evolutionary Microbiology. 66, 5379–5383 DOI 10.1099/ijsem.0.001524 . Oulghazi S., Cigna J., Lau Y.Y., Moumni M., Chan K.G., Faure D. (2019). Transfer of the waterfall source isolate Pectobacterium carotovorum M022 to Pectobacterium fontis sp. nov., a deep-branching species within the genus Pectobacterium. Int J Syst Evol Microbiol. 69(2): 470-475. doi: 10.1099/ijsem.0.003180. . Oulghazi S., J. Pédron, J. Cigna, YY. Lau, M. Moumni, F. Van Gijsegem, KG Chan, D. Faure (2019). Dickeya undicola sp. nov., a novel species for pectinolytic isolates from surface waters in Europe and Asia. Int J Syst Evol Microbiol. doi: 10.1099/ijsem.0.003497. . Portier P., Pédron J., Taghouti G., Fischer-Le Saux M., Caullireau E., Bertrand C., Laurent A., Chawki K., Oulgazi S., Moumni M., Andrivon D., Dutrieux C., Faure D., Hélias V., Barny MA. (2019). Elevation of Pectobacterium carotovorum subsp. odoriferum to species level as Pectobacterium odoriferum sp. nov., proposal of Pectobacterium brasiliense sp. nov. and Pectobacterium actinidiae sp. nov., emended description of Pectobacterium carotovorum and description of Pectobacterium versatile sp. nov., a novel species isolated in various

15 Euphresco project report

geographic location from water streams and symptoms on diverse plants. Int J Syst Evol Microbiol. Int J Syst Evol Microbiol 69:3214–3223. DOI 10.1099/ijsem.0.003611 . Raoul des Essarts Y., Cigna J., Quêtu-Laurent A., Caron A., Munier E., Beury-Cirou A., Hélias V., Faure D. 2015. Biocontrol of the potato blackleg and soft-rot disease caused by Dickeya dianthicola. Appl Environ Microbiol 82:268–278. doi:10.1128/AEM.02525-15 . Raoul des Essarts Y., Pédron J., Blin P., Van Dijk E., Faure D., Van Gijsegem F. (2019). Common and distinctive adaptive traits expressed in Dickeya dianthicola and Dickeya solani pathogens when exploiting potato plant host. Environ Microbiol. 21(3): 1004-1018. doi: 10.1111/1462-2920.14519. . Royet K., Parisot N., Rodrigue A., Gueguen E., Condemine G. (2019). Identification by Tn- seq of Dickeya dadantii genes required for survival in chicory plants. Molecular Plant Pathology, 20:287-306. . Sarfraz S., Riaz K., Oulghazi S., Cigna J., Talib Sahi S., Habibullah Khan S., Faure D. (2018). Pectobacterium punjabense sp. nov., isolated from blackleg symptoms of potato plants in Pakistan. Int J Syst Evol Microbiol. 68(11):3551-3556 . Tsror, L., Lebiush S., Erlich, O., Galilov, I., Chalupowicz, L., Reuven, M., Dror, O., Manulis- Sasson, S. (2019). First report of latent infection of Malva nicaeensis caused by Pectobacterium carotovorum subsp. brasiliense in Israel. New Disease Reports 39, 4 (doi.org/10.5197/j.2044-0588.2019.039.004) . Van der Wolf, J.M., De Haan, E.G., Kastelein, P., Krijger, M., De Haas, B.H., Velvis, H, Mendes, O., Kooman-Gersmann, M. and Van der Zouwen, P.S. (2016). Virulence of Pectobacterium carotovorum subsp. brasiliense on potato compared with that of other Pectobacterium and Dickeya species under climatic conditions prevailing in the Netherlands. Plant Pathology DOI: 10.1111/ppa.12600. . Zijlstra, C., Groenenboom–De Haas, L., Krijger, M., Verstappen, E., Warris, S., De Haan, E., and Van Der Wolf, J. (2019). Development and evaluation of two TaqMan assays for generic detection of Dickeya species. European Journal of Plant Pathology, 1-6.

16 Euphresco project report

4. Open Euphresco data None.

17 Euphresco project report

Euphresco III “Dickeya & Pectobacterium meeting”

22 - 24 November 2015, Gdansk, Poland

1 Local organizer: Prof. PhD E. Lojkowska (IFB UG & MUG, Gdansk, PL) PhD Robert Czajkowski, PhD Marta Potrykus, PhD Wojciech Sledz, PhD Małgorzata Waleron Agata Motyka, Sabina Żołędowska Project coordinators: PhD Maria Bergsma (NVWA, NL) and PhD Jan van der Wolf (WUR, NL)

3 The effect of soil temperature and moisture on survival of Pectobacterium carotovorum subsp. brasiliense in fallow soil: Preliminary results

J.E. van der Waals, N. Laughton & L. Nhukarume

Department of Plant Science, University of Pretoria, Private Bag X20, Hatfield, Pretoria, 0028, So- uth Africa

Monitoring of pathogen levels in soil is an important tool for disease management and understanding pathogen ecology. One of the most important pathogens in the South African potato industry is Pectobacterium carotovorum subsp. brasiliense (Pcb), the causal agent of soft rot and blackleg of potatoes. Although many articles have stated that pectinolytic bacteria are not able to survive for extended periods of time in soil in the absence of a host, none of these studies were conducted on Pcb. The aim of this experiment is thus to determine how long and under which environmental conditions Pcb is able to survive in fallow soil. Pilot pot trials using a clay-loam soil were set up at three different temperatures, 10, 20 and 30˚C. The soil in each of the temperature treatments was maintained at two different moisture conditions i) dry (no watering at all) and ii) wet (satu- ration point). A 108 cfu/ml suspension of a GFP-tagged Pcb isolate was used to inoculate the soil. Soil samples were taken regularly after inoculation from each temperature / moisture treatment to determine survival of the bacterium in the soil. A dilution series was made of each soil samples and plated out onto selective medium. Resultant colonies were counted and viewed under luorescent light to conirm the presence of the GFP-tagged isolate. The concentration of bacteria in samples was determined also determined by qPCR. Preliminary results showed that four to six weeks after inoculation Pcb was no longer detectable in soil by conventional plating out. However, bacteria were still detected by qPCR up for ive months after inoculation of the soil. This is pro- bably due to ampliication of non-living bacterial DNA in the soil. Pcb survived longest in the wet soil and at warmer temperatures than in dry soil or at 10˚C. This trial conirms results of various other studies on survival of pectinolytic bacteria, which indicate that these bacteria are unable to survive in soil for extended periods of time in the absence of a host. A second trial is currently underway to verify the results of the irst.

1 Monitoring of imported and national seed lots in the control of pectinolytic bacteria in the Swiss potato Industry (2013-2015)

Patrice de Werra and Andreas Keiser

School of Agricultural, Forest and Food Sciences (HAFL), Bern University of Applied Sciences, Swi- tzerland

During recent years, possibilities of minimizing the impact of blackleg in Swiss seed potato production have been discussed. Complementary to the oficial seed potato certiication scheme based on visual inspection in the ield, a reliable routine method to assess the health status of seed tuber lots is being sought. In an on-farm project conducted on behalf of the Swiss Federation of Seed Potato Growers (swisssem) from 2013 to 2015, the relationship between seed infection and blackleg incidence in the ield was investigated. PCR/qPCR tests were performed to detect latent infections in seed lots by the causal agent of blackleg. The focus was on Dickeya sp. (Dsp), Pectobacterium atrosepticum (Patr), Pectobacterium wasabiae (Pwas) and Pectobacterium carotovorum subsp. brasiliense (Pcbr), which are the most problematic pectinolytic bacteria encountered recently in Swi- tzerland. Having been in steep decline in Switzerland since 2012, Dsp and Patr were almost absent in 2015, in both seed latent infection and in the ield. Since 2013, Pcbr has gained in importance and was still the most problematic agent of blackleg this year. Although the reliability and usefulness of PCR/qPCR tests as a diagnostic tool seem to be acceptable for Dsp and Patr, this is not the case for Pcbr. This bacterium was found in latent infections in a very large part of the seed lots analyzed, but was not consistently seen in the ield. This led to an increased number of false positive analyses, which weakened the reliability of the test for this bacterium.

2 Understanding the epidemiology of Pectobacterium in-field spread and blackleg disease

Emma Campbell1, Sonia Humphris1, Greig Cahill2, Yvonne Nova2, Gerry Saddler2, John Elphinstone3, Stuart Wale4 and Ian Toth1

1James Hutton Institute, Invergowrie, Dundee DD2 5DA 2Science and Advice for Scottish Agriculture (SASA), Edinburgh EH12 9FJ 3Fera Science Ltd., York YO41 1LZ 4Scotland’s Rural College, (SRUC), Aberdeen AB21 9YA

Experimental plots were used to track the movement of a streptomycin resistant strain of Pec- tobacterium atrosepticum (Pba) from an infected central zone to healthy neighbouring pla n t s . During the growing season, leaf, stem, root and tuber samples were taken from plants at radiating points from the central infector zone and tested for the presence of the marked strain and naturally occurring isolates. At the end of the growing season samples were taken from the stem of all plants showing blackleg symptoms and the pathogen present was identiied. After haulm destruction the tubers designated to be sampled for assessment of infection were hand dug from the plot and tested to determine whether Pectobacterium infection was systemic (found in the vascular tissue of stolon end), or as lenticel infection in tuber peel. Our results conirmed movement of the streptomycin resistant Pba strain from infected to healthy plants. However, the data also revealed that natural isolates of Pectobacterium, including but not only Pba, were more prevalent than Pba 1039 strepR in terms of both contamination and in causing blackleg disease. Pba 1039 strepR and natural isolates of Pectobacterium were detected in both the peel and stolon end of the harvested tubers.

3 Lytic bacteriophages against soft rot Enterobacteriaceae – isolation, characterization and future perspective

Robert Czajkowski1, Z. Ozymko1, V. de Jager2, E. Lojkowska1,

1 Department of Biotechnology, Intercollegiate Faculty of Biotechnology UG and MUG, Kladki 24, 80-822 Gdansk, Poland 2 Netherlands Institute of Ecology (NIOO-KNAW), Wageningen, the Netherlands

Pectobacterium spp. and Dickeya spp. are necrotrophic bacterial pathogens of many important crops worldwide. The effective strategies to control pectinolytic bacteria have not yet been developed. Consequently, the management of Pectobacterium spp. and Dickeya spp. is based mainly on the exclusion of infected plant material and the use of hygienic practices during potato cultivation and in storage. This study reports on the isolation and characterization of broad host lytic bacteriophages able to infect the dominant Pectobacterium spp. and Dickeya spp. affecting potato in Europe viz. Pectobacterium carotovorum subsp. carotovorum (Pcc), P. wasabiae (Pwa) and Dickeya solani (Dso) with the objective to assess their potential as biological disease control agents. The lytic bacteriophages were isolated from potato samples collected in different potato ields in Poland. The phages were characterized for features that are potentially important for successful biological control applications and bacteriophages’ stability in the environment. Transmission electron microscopy was used to study bacteriophage morphology. In the interaction studies, the phages were characterized for optimal multiplicity of infection, the rate of adsorption to the bacterial cells, the latent period and the burst size. The selected bacteriophages were also genotypi- cally characterized with RAPD-PCR and RFLP techniques. The structural proteomes of selected phages were obtained by fractionation of phage proteins by SDS-PAGE. Phage protein identiication was performed by liquid chromatography-mass spectrometry (LC-MS) analysis. Pulsed-ield gel electrophoresis (PFGE), genome sequencing and comparative genome analysis were used to gain knowledge of the length, organization and function of the genomes.

4 Dickeya solani, the game changer in the 2015 cropping season potato blackleg outbreaks in north Finland

Yeshitila Degefu and Holappa, O.

Natural Resources Institute Finland (LUKE), Green Technology, Pa- avoHavaksen tie 3, FI-90014 University of Oulu, Finland

During the last three decades, the etiology of blackleg and soft rot in Finland has shifted from a serotype of one species - Pectobacterium atrosepticum to multiple species. To date Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. brasiliense, Pectobacterium carotovorum, Pecto- bacterium wasabiae and Dickeya solani are frequently detected from potato with typical blackleg symptoms. Furthermore, it is intriguing to observe a signiicant number of samples with typical blackleg where none of these currently known potato blackleg and soft rot bacteria are detected. A circumstance tempting to suggest “unknowns” to the list and a revisit to the etiology of the disease complex. The Dickeya and Pectobacterium species listed above are known to be adapted to different temperature optimums and nowadays disease has become a likely phenomenon in Fin- land irrespective of the prevailing summer weather- warm or cool. According to the 2015 weather statistics report by Finnish Meteorological Institute (http://en.il- matieteenlaitos.i/press-release/98978129) June was unusually cold 1-2 degrees colder than usual with exceptionally high precipitation especially in North Finland. July was exceptionally cold. The monthly average temperature in July remained below the long-term average in the whole country, varying from just over 15°C in southern parts of the country to less than 11°C in northern Lapland. A July as cool as this year occurs in the region on average once in 30 years. After two months of colder-than-average weather, August turned out to be warmer than average across the whole country. The average temperature for the whole country was 14.9°C, which was 1.4°C above normal levels. The month also saw a total of 15 hot days (where temperature rose over 25°C). The last time that August was the hottest month of the summer was in 2006. Until the end of July, no blackleg symptoms were reported by farmers in the region. Just in the irst and second week of August, severe and rapidly spreading blackleg outbreaks were reported from two separate ields of the same variety and seed background. The game changer was D.solani. Further characterization of the outbreaks and analysis of weather data will be discussed.

5 Effect of lenticular and vascular infection with Dickeya spp. on blackleg symptom expression in potato fields

Brice Dupuis, Gaétan Riot, Isabelle Kellenberger and Santiago Schaerer

Agroscope, Institute for Plant Production Sciences IPS, 1260 Nyon 1, Switzerland

Potato blackleg is known to be a seed borne disease caused by pectinolytic bacteria belonging to the genera Pectobacterium and Dickeya. The seed tuber can be infected through the lenticels and/or through the vascular system of the plant. Lenticelar inoculation is broadly used to assess the aggressiveness of Pectobacterium and Dickeya strains and is also commonly used to assess the susceptibility of the cultivars to blackleg. Nevertheless, little is known about the relevance of those results to assess the risk of transmission of the inoculum to the progeny tubers, through the vascular system of the plant. Field experiments were set up in Switzerland to answer this question: 4 to 19 cultivars were inoculated with one D. dianthicola strain (8823) and cv. Agria was inoculated with 5 to 6 Dickeya strains. Four hundred seed tubers were inoculated by soaking, planted in the ield (year 1) and then, blackleg symptoms were assessed 12 to 16 weeks after planting. After harvest, 100 tubers from each plot were stored at 4°C and planted the following year and blackleg symptomatic plants were counted (year 2). This two year trial was repeated twice with respectively 5 and 6 Dickeya strains and thrice with respectively 5, 7 and 19 potato cultivars. For each trial, we tested the correlation between the expression of blackleg symptoms in year 1 and year 2. The results revealed that the correlation is better for aggressiveness trials (R-squared of 0.51 and 0.38) than for cultivar susceptibility studies (R-squared of 0.07, 0.06 and 0.21). This work indicates that the results of trials using lenticelar inoculation should be interpreted with caution, since they are not taking into account the infections of the progeny tubers through the vascular system of the plant. The results of trials using lenticelar inoculation are giving a partial indication of the susceptibility of one potato cultivar to various pectinolytic bacteria strains, but can reach to wrong conclusions if the objective of the trial is to distinguish the susceptibility of different cultivars to those bacteria.

6 Spreading of soft rot bacteria with nematodes and winter flies

Minna Pirhonen, Nykyri J. & Pasanen M.

Department of Agricultural sciences, University of Helsinki, Finland.

We have investigated the role of free-living nematodes and winter crane lies multiplying in storage rooms in the spreading of soft rot bacteria. Soft-rot enterobacteria were able to withstand nematode grazing, colonize the gut of Caenorhabditis elegans and subsequently disperse to plant material while remaining virulent. Two nematode species were also isolated from a rotten pota- to sample, and one of these isolates (Pristionchus sp.) was shown to be able to disperse soft-rot enterobacteria to plant material (Nykyri et al. 2013). The winter crane lies were collected from storage rooms and identiied as Trichocera maculipennis. This species is known of antropogenic associations - in localities of cold climate it chooses relatively warm shelters, as cellars and caves, and often migrates with humans. No soft rot bacteria were identiied in the adult winter crane lies collected from potato storage rooms. However, when the lies were exposed to potato tuber tissue containing soft rot bacteria, they were able to transfer the bacteria onto surface sterilized potato tubers. Furthermore, when the exposed potato tubers were incubated in rot-promoting conditions, they rotted faster than the control tubers. In conclusion, nematodes may have a role in the short-distance spreading of soft rot bacteria in soil, whereas the winter crane lies may spread the bacteria in potato storage rooms to clean harvest if rotten tubers are present in the same storage. Nykyri et al. 2013 Plant Pathology 63:747-757

7 Characterizing genetic resources to control the potato pathogen complex species belonging to Pectobacterium and Dickeya

Hélias Valérie 1, 2, 4, Quêtu-Laurent Angélique 1, 2, 4, Pellé Roland 3, 4, Le Hingrat Yves 1, 4, Marhadour Sylvie 1, 3, 4, Andrivon Didier 2, 4, Kerlan Marie-Claire 3, 4

1 FN3PT/RD3PT, French Federation of Seed Potato Growers- Research, Development, Promotion of Seed Potatoes, 43-45 rue de Naples, F-75008 Paris, France 2 INRA, UMR1349 IGEPP, F-35653 Le Rheu Cedex, France 3 INRA, UMR1349 IGEPP, Keraiber, F-29260 Ploudaniel, France 4 UMT Innoplant, UMR1349 IGEPP, F-35653 Le Rheu Cedex

Pectobacterium and Dickeya are the two bacterial genera involved in blackleg and soft rot symptoms in potato. They cause major damage and costly losses in Europe and beyond. The use of genetic resistance constitutes an interesting prospect to control these diseases, which currently relies exclusively on prophylactic measures. Progenitors with resistance to P. atrosepticum have been identiied by INRA in the late 1990’s, made available to the French potato breeders and introduced in useful genetic material (cultivars or hybrids) [1] [2]. However, annual surveys and diversity studies conducted by FN3PT-RD3PTin France over the last decade have shown evolutions in the prevalence and distribution of the species/subspecies complex responsible for soft-rot and blackleg [3]. One part of the National Rese- arch Program on Pectobacterium and Dickeya (FN3PT-RD3PT-INRA) aim at evaluating the performance of the resistance sources identiied some years ago against P. atrosepticum to control the other members of this pathogenic bacterial complex. A collection of 24 hybrids from different genetic backgrounds was tested against representative strains of the main taxa responsible for soft rot and blackleg. Ten of these clones showed very low soft rot severity in front of two to ive species/subspecies, and 9 additional clones showed speciic resistance to one of these species. Experiments to test the resistance level of clones against blackleg (stem infections) will be conducted. These promising results open new hopes for the sustainable control of the soft rot/backleg complex in potato.

[1] Andrivon et al., 2003. Amer J Potato Res 80: 125-134 [2] Pasco et al., 2006. Potato Res 49 : 91-98. [3] Hélias et al., 2014, 19th Triennial Conference of the EAPR, Brussels : p.110

8 The metabolic phases of the soft-rot disease

Nicole Hugouvieux-Cotte-Pattat

CNRS UMR5240 Microbiologie Adaptation et Pathogénie, Université de Lyon, Uni- versité Claude Bernard Lyon 1, INSA de Lyon, F-69621Villeurbanne.

The soft-rot disease caused by Dickeya dadantii is a dynamic process usually divided in two main phases. Bacteria penetrate their host using wounds or natural openings. During the irst hours, bacteria reside latently in the plant intercellular space without provoking any symptoms. This “asymptomatic phase” is followed by a short transition to the “symptomatic phase” corresponding to appearance of the soft-rot symptoms. Metabolomics (13C-NMR spectroscopy) was used to analyze the metabolic activity of the pathogen. After infection of chicory leaves by D. dadantii 3937, the asymptomatic and the symptomatic phases corresponded to 1-8 hours and 12-24 hours, respectively. A kinetic analysis, with 4 h intervals, was performed to follow the infectious phases. The modiications observed during infection gave a direct image of the bacterial metabolism. During the asymptomatic phase, bacteria began to assimilate some easily metabolizable plant soluble sugars. A controlled repression of aggressive factors allows the bacteria to multiply without being recognized by their host. The transition step corresponds to a strong metabolic change including induction of pectate lyase production. The symptomatic phase corresponds to the expansion of soft-rot due to the pectate lyase burst. Bac- teria multiply to large population sizes by obtaining more nutrients through the degradation of plant cell wall polysaccharides.

9 Whole-genome comparison-based classification of the soft rot enterobacteriaceae

Leighton Pritchard1, Sonia Humphris1, Rachel Glover2, John Elphinstone2 and Ian Toth1

1James Hutton Institute, Invergowrie, Dundee DD2 5DA 2Fera Science Ltd., York YO41 1LZ

Effective detection and diagnosis of the soft rot Enterobacteriaceae (SRE) is dependent on the ability to classify pathogenic isolates accurately. Diagnostics and classiication are made more dificult by the inluence of horizontal gene transfer on phenotype, and historically complex and sometimes inaccurate nomenclatural and taxonomic assignments that persist in strain collections and online sequence databases. We present novel whole-genome classiications of the SRE using average nucleotide identity (ANI), illustrating inconsistencies between the established taxono- mies and evidence from completely sequenced isolates

10 Mechanism of antimicrobial activity of Pseudomonas sp. P482 against soft rot pathogens: a study aided by genome mining

Dorota M. Krzyżanowska, Adam Ossowicki, Magdalena Rajewska, Tomasz Maciąg & Sylwia Jafra

Laboratory of Biological Plant Protection, Intercollegiate Faculty of Biotechnology UG&MUG, Kładki 24, 80-822 Gdansk, Poland

Pectobacterium and Dickeya are plant-pathogenic bacteria causing soft rot disease of many vegetables and ornamental plants. Despite serious economic losses caused by these bacteria, the means to ight these pathogens are limited. Pseudomonas sp. P482, a Gram-negative bacterium originating from the rhizosphere of tomato, is able to inhibit the growth of various species of Pectobacte- rium and Dickeya in a plate assay and to attenuate plant tissue maceration caused by the selected strains of these pathogens. To reveal the mechanisms involved in the antagonism between Pseudomonas sp. P482 and the soft rot pathogens, a transposon mutagenesis and a genome mining approach in combination with the site-speciic mutagenesis were applied. The genome sequence of Pseudomonas sp. P482 was obtained, automatically annotated and subjected to data mining in search for secondary metabolite biosynthetic clusters. The search was performed both manually, with the blastp tool, and automatically, with the antiSMASH 2.0 software. Five genes were selected based on the in silico study, each representing one biosynthetic cluster predicted by the antiSMASH 2.0 as a result of a default search against its internal database. Knock out of these genes showed that they are not responsible for the antimicrobial activity of P482 against the soft rot bacteria. A more unrestricted antiSMASH 2.0 search based on Pfam domain probabilities revealed 18 more clusters designated as “hypothetical”. A transposon mutant of P482 not inhibiting the growth of Dickeya spp. in vitro, carries an insertion in one of these “hypothetical” clusters. Thus, genome mining for secondary metabolite clusters is a powerful approach although when novel mechanism are at stake, it should be used in combination with other methods

11 Implementation of research results on Erwinia into the seed production in The Netherlands.

Kees Kristelijn

HZPC, Edisonweg 5, 8501 XG Joure

In the Netherlands, after the Erwinia problems in the early 2000’s, a survey was held under 300 seed potato growers. After a scan of the problems, and the answers of the farmers in the survey, a project with all major stakeholders was started in 2005. During the periods 2005 – 2009 (“Bac- terievrije pootgoedteelt”) and 2009 – 2013 (“Deltaplan Erwinia”) research was done on almost all aspects of growing seed potatoes. All this research lead to a list of do’s and don’ts which had to be implemented within the process of growing seed potatoes. A group of 8 companies , with more than 90% of the total acreage of seed potato production in the Netherlands, started a new project called Deltaplan Erwinia 2.0. HZPC started to work with the results of the irst project in 2009. Ad- justments in advice’s to growers, and testing of basic seed material, have lead to less problems in degrading and rejection of seed lots in the last years in the Netherlands. This, despite the growing concern about the development of P.c. subsp. Brasiliense. In the years after 2009 the development of Erwinia in the different HZPC seed lots was monitored. In my presentation I will highlight some results of the irst year of Deltaplan 2.0 and go in to more detail in the way HZPC acts on the implementation of research results on Erwinia into the seed potato production in the different seed production area’s in Europe.

12 Characterization of pectinolytic bacteria isolated in potato in Poland and Norway in 2013

Renata Lebecka1, A. Grupa1, M. Wiken Dees2, J. Perminow2, R. Nærstad2, M. B. Brur- berg2, A. Motyka3, S. Żołędowska3, W. Śledź3, M. Potrykus3, M. Golanowska3, R.Czaj- kowski3, E. Łojkowska3

1 Plant Breeding and Acclimatization Institute – National Research Institute, Mlochow Division, Platanowa 19, 05-831 Mlochow, Poland 2 Norwegian Institute of Bioeconomy Research, Høgskoleveien 7, NO-1431, Ås, Norway 3 Intercollegiate Faculty of Biotechnology UG&MUG, Kładki 24, 80-822, Gdańsk, Poland

Bacteria belonging to Pectobacterium and Dickeya spp. cause soft rot diseases of a great variety of crops and ornamentals worldwide. In the last 5 years potato losses caused by soft rot have increased signiicantly and it appears that warmer climate escalate the disease development. We investigated the population structure of pectinolytic bacteria (Dickeya/Pectobacterium species) in Poland and Norway. The survey was conducted in 2013, and a representative selection of species and isolates from both countries, were examined in detail. From Poland, 17 Pectobacterium wasabiae (Pw), 14 P. atrosepticum (Pba), 9 P. carotovorum subsp. carotovorum (Pcc), and 2 - Dickeya solani (Ds) were selected. From Norway, 21 Pcc and 18 Pa isolates were selected. Single isolates of Pw and Ds were found. The isolates were characterized for several phenotypic traits: the aggressiveness on potato tubers, production of cellulases, pectinases, proteases, siderophores, motility, and growth in the presence of high salt concentration (5% NaCl). A maceration test on tubers of the susceptible potato cultivar Irys, at 26oC, showed high variability in aggressiveness within each bacterial species. The isolates from Norway were in average more aggressive than the Polish ones. The Pcc isolates were signiicantly more aggressive than the Pba isolates, and these were more aggressive than the Pw isolates. Polish strains of Pba, Pcc, Pwa and Ds did not differ signiicantly from Norwegian strains in other analyzed phenotypic traits. Individual isolates exhibited differences especially in siderophore, protease production and motility. Only three of the analyzed Pba strains produced proteases. The prediction of bacterial species based on discriminant analysis of preliminary data of all phenotypic traits ranged from 0.667 (for Pwa) to 1.0 (for Ds). The work is in progress within Polish-Norwegian POTPAT project.

13 Mass spectrometry in microbiological research.

Katarzyna Macur, Paulina Czaplewska

Laboratory of Mass Spectrometry, Core Facility Laboratories, Intercollegiate Faculty of Biotechnology UG-MUG, ul. Kładki 24, 80-822 Gdańsk, Poland

Mass spectrometry (MS) is a powerful tool to investigate microbial world. New developments in MS instrumentation and analytical worklows have been contributing to wider application of this technique in different areas of microbiology, such as taxonomic identiication or microbial interactions [1]. The presentation will focus on different applications of mass spectrometry equipment of the Laboratory of Mass Spectrometry, supported by the FP7 MOBI4Health project, in microbial protein research performed at the at the Intercollegiate Faculty of Biotechnology UG-MUG. The examples will include application top-down and bottom-up proteomic approaches for identiication of intact proteins and protein digests, respectively. The SILAC-based protein identiication and quantiication of differently treated Candida albicans cells will also be described. Apart from presently used at the IFB UG-MUG MS worklows, new applications available on our instrumentation of particular interest for microbiological studies will be shown.

[1] T. Luzzatto-Knaan, A.V. Melnik, P.C. Dorrestein: Mass spectrometry tools and worklows for revealing microbial chemistry. Analyst 2015, 140:4949.

14 Biodiversity of Dickeya spp. isolated from potato plants and water sources in temperate climate

Marta Potrykus1, M. Golanowska1, W. Sledz1, S. Zoledowska1, A. Motyka1, A. Kolodziej- ska2, J. Butrymowicz2 & E. Lojkowska1

1 Laboratory of Plant Protection and Biotechnology, Intercollegiate Faculty of Biotechnology of University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822 Gdansk, Poland. 2The Central Laboratory of the State Plant Health and Seed Inspection Service, Zwirki i Wigury 73 87-100 Torun, Poland

Bacteria from the genera Dickeya (formerly Erwinia chrysanthemi) and Pectobacterium (formerly Erwinia carotovora) are the agents of blackleg and soft rot disease on many important crops. In 2005, D. solani was isolated for the irst time in Poland from a symptomatic potato plant. This motivated us to study the presence and diversity of Dickeya spp. in potato ields and water sources (including surface waters near potato ields, and water from potato processing facilities and sewage plants) in temperate regions of Poland (Central Europe). Only D. dianthicola and D. solani were isolated from symptomatic potatoes, and only D. zeae and D. chrysanthemi were isolated from water sources. The Dickeya spp. isolated from potato formed a relatively homogenous group, while those from water sources were quite diverse. To our knowledge, this is the irst comprehensive characterization of Dickeya spp. isolated from regions with a temperate climate in Central Europe.

15 Survey of grower practice and identiϐication of contributory factors which may be causing an increase in blackleg occurrence in Scotland

Gerry Saddler1, Triona Davey1 & Claire Hodge2

1 SASA, Edinburgh EH129FJ, Scotland, UK 2 AHDB Potatoes (Edinburgh), Scottish Office Rural Centre West Mains, Newbridge Midlothian, EH28 8NZ, Scotland, UK

Blackleg remains the biggest cause of down-grading and rejection in the Scottish Seed Potato Clas- siication Scheme. Currently, control is reliant on initiating production from disease free planting material, industry good practice and the implementation of the Classiication Scheme itself. In Scotland, healthy planting material arises from disease-tested micro-plants produced by SASA, which are multiplied at 8 mini-tuber production facilities, under controlled conditions, across the country. Field grown potatoes are initially produced as pre-basic (PB) seed by 28 registered growers, where a zero-tolerance for blackleg (and other faults) is applied. Crop inspections during every year of ield multiplication serve to limit/remove heavily diseased crops from the production chain. Up until 2007 this approach was judged to be effective however more recently blackleg incidence has risen steadily, peaking in 2011 to levels not seen for more than 20 years. In addition, post -harvest tuber testing of PB stocks has shown that some crops can become infected with Pectobac- terium atrospeticum (in the absence of symptoms) as early as FG1. It is also clear that by looking at the national picture from ield inspections returns of basic crops, the incidence of blackleg generally increases with each subsequent ield generation. The purpose of this study was therefore to investigate what inluence, if any, social changes/industry practices may have on blackleg incidence with a view to identifying contributing factors, should they exist. The survey initially focused on Scotland’s 28 PB growers (alongside 1 PB grower each from England and Northern Ireland) and was based on one-to-one interviews in which a series of questions around social, inancial and economic change were addressed. Results from this part of the study highlight possible contributory factors which may in part be contributing to the rise in blackleg incidence. The next stage in the process will be described from which, it is hoped, a reined approach to blackleg control will be derived.

16 Dickeya solani and Pectobacterium spp. in Israel

Leah Tsror

Department of Plant Pathology and weed Research, Agricultural Research Organization (ARO), Gilat Research Centre, MP Negev 85280, Israel.

Potato is being cultivated in two major seasons in Israel; seed lots are imported from Europe for the spring each year whereas for the fall-winter season the growers use their own seeds (produced in spring under inspection of PPIS). Spring begins with low temperatures followed by increase, while the fall starts with high temp which decrease later. Pectinolytic bacteria cause economic losses with symptoms including pre-emergence tuber rot, blackleg and wilt. Although the pectinolytic bacteria are transmitted mostly by latent infection of seed tubers, disease symptoms vary with climate. Dickeya solani (Ds), Pectobacterium carotovorum (Pc), P. c. brasiliense (Pcb) and P. wassabiae (Pw) can grow at low and high temperatures with higher maximal growth temperature compared with D. dianthicola or P. atrosepticum (Pa). Development and expression of the disease in ield may be particularly favored under warm-climate conditions. The protocol for detecting Dickeya-latent infection in seed lots by enrichment RT-PCR was used to detect also other Pectobacterium spp. In 2015, 77 commercial imported seed lots were checked for latent D. solani and Pectobacterium spp. infections. Field surveys indicated that 69% of seed lots that were Ds-negative had no disease symptoms in ields (-L/-F), 19% were Ds-positive lots with ield symptoms (+L/+F), 9% of seed lots were Ds-negative but had symptoms (false negative; -L/+F) and 3% were false positive (+L/-F). In a ield trial conducted in 2015, seed tubers of rando- mly selected imported seed lots (10 lots of cv. Sifra, 6 Mozart, one of each cvs. Winston, Marabel, Rodeo, Canberra) were planted at Gilat (4 reps X 50 tubers). Wilt and blackleg symptoms were weekly monitored and the presence of pectinolytic bacteria was checked in seed tubers, plants and progeny tubers. In the selected seed lots, Ds and Pcb were detected in 75% and 55%, respectively. At the end of the experiment all sampled plants were contaminated with Ds, and 45% had Pcb. In progeny tubers, 75% were infected with Ds, and 70% with Pcb. Ware potatoes were recently exported to Jordan but only after lab test for Ds. Out of 39 tested lots 11 were Ds-positive (28%) and therefore rejected (among them: Sifra - 20 lots with 7 rejected; Winston 8 lots, 2 rejected; Pa- namera 2 lots, one rejected). The presence of Ds and Pcb in imported seed material directly affect yields in the spring crop in Israel, as well as on export of ware potato produced in this season in addition to the fall crop which might be also affected. Pathogenicity of Pcb isolates using the pota- to tuber maceration test was evaluated and further characterization will be carried out.

17 Comparison of Dickeyas expression profiles in macerated tubers and Dickeya solani diversity

Jacques Pédron1, Yannick Raoul des Essart2,3, Slimane Khayi2,4, Valérie Hélias3, Denis Faure2 and Frédérique Van Gijsegem1

1 iEES Paris (Institute of Ecology and Environmental Sciences), INRA-UPMC Université Paris 06, UMR 1392, 7 quai Saint-Bernard F-75005, Paris, France 2 Institut for Integrative Biology of the Cell (I2BC), CNRS-CEA-Université Paris-Sud, Saclay Plant Sciences, UMR9198, Avenue de la Terrasse, 91198 Gif-sur-Yvette cedex, France 3 Research, Development, Promotion of Seed Potatoes - French Federation of Seed Potato Growers (RD3PT-FN3PT), 43-45 Rue de Naples, 75008, Paris, France 4 Université Moulay Ismaïl, Faculté des Sciences, Département de Biologie, Meknès, Maroc

Comparison of the complete genome sequence of one D. solani and one D. dianthicola strains, both isolated from diseased potato in France, revealed a high synteny between these genomes. Both genomes only harbour a few hundred genes present in only one of the two species. Interestingly, these species-speciic genes are often clustered in genomic regions, frequently associated with mobile elements in D. dianthicola. Most of these genomic regions regroup genes that are predicted to be involved in metabolism/transport as well as regulatory genes. Comparison of gene expression proiles after tuber infection reveals that the expression of several of these species-speciic genes is modulated in planta as compared to in vitro bacterial growth pointing to an involvement in plant-bacteria interactions. A different modulation of several genes that are common to both species was also observed in planta. This indicates speciicities in gene regulation between both species and highlights the importance of expression analysis to unravel the complete diversity of related strains/species in their interaction with their hosts. The D. solani diversity was analysed by comparing the complete genome sequence of D. solani strain 3337 with 19 other D. solani genomes of strains isolated in different years, from various geographical locations and from different hosts. This population genomic analysis highlighted an unexpected variability among D. solani isolates. Indeed, in addition to scattered SNP/InDel variations, replacing and additive horizontal gene transfers (HGT) were observed by inter-species in- trogression of D. dianthicola genomic regions and plasmid acquisition. Furthermore, this analysis led to the characterization of two distinct sub-groups within the D. solani species differing to each other by variations (mainly SNP/InDels) in about one half of the genes. Replacing HGTs were also observed between the two D. solani sub-groups.

18 Virulence of Pectobacterium and Dickeya species in potato under climate conditions in Western Europe

Jan van der Wolf1, E.G. de Haan2, P. Kastelein1, M. Krijger1, B.H. de Haas1, H. Velvis1, O. Mendes1 & P.S. van der Zouwen1

1 Plant Research International, P.O. Box 16, 6700 AA Wageningen, The Netherlands.

2 Dutch General Inspection Service for Agricultural seed and seed potatoes (NAK), P.O.Box 1115, Randweg 14, 8300 BC Emmeloord, The Netherlands

Dickeya and Pectobacterium strains, isolated from potato or from surface water in the Netherlands were tested in ield experiments for their virulence in potato, Seed potatoes were vacuum-iniltra- ted and planted in 2013 and 2014 in randomized blocks in a clay soil in the Netherlands. Blackleg and slow wilt symptoms were recorded during the growing season. In both seasons, inoculation with strains of Pectobacterium atrosepticum and P. carotovorum subsp. brasiliense resulted in the highest disease incidences of between 75 and 95 percent of the plants that emerged. Inoculation with strains of D. solani and P. wasabiae resulted in disease incidences between 5 and 25 percent. Hardly any or no disease was observed in the treatments with four strains of P. c subsp. carotovorum strains, a strain of D. dianthicola and the water control. Co-inoculations of seed potatoes with a strain of P. c. subsp. brasiliense and D. solani gave a similar disease incidence as inoculation with P. c. subsp. brasiliense only. However, co-inoculation of P. carotovorum subsp. brasiliense with P. wasabiae decreased the disease incidence compared to inoculation with P. c. subsp. brasiliense. The presence of pathogens in progeny tubers of plants was conirmed with enrichment-TaqMan assays and for some treatments with dilution plating followed by a colony-TaqMan assay. For this, speciic TaqMan assays were developed and evaluated for detection of P. c. subsp. brasiliense and P. wasabiae whereas a TaqMan for detection of P. atrosepticum previously developed previously at FERA (UK) was evaluated.

19 Plant phenolic acids affect the virulence of Pectobacterium spp. via quorum-sensing regulation

Janak Raj Joshi1, 2, Alexander Lipsky2, Shaked Yariv2 and Iris Yedidia2*

1 D e p a r t m e n t o f P l a n t P a t h o l o g y a n d M i c r o b i o l o g y a n d t h e O t t o Wa r b u r g M i n e r v a C e n t e r f o r Agricultural Biotechnology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel 2 Department of Plant Sciences, Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel * Correspondence. E-mail: [email protected]

The virulence strategy of Pectobacteria is based on the secretion of exoenzymes that degrade the cell wall of their hosts, providing nutrients to the bacteria on the one hand, but conversely expo- sing the bacteria to plant defense compounds on the other. To better understand the effects of these plant-derived antimicrobial molecules, we have screened phenolic acids and polyphenols, for their ability to affect virulence in several Pectobacteria. The results revealed a signiicant effect of some phenolic compounds on virulence determinants such as motility, bioilm formation and extracellular enzyme activities. Moreover, short exposure to some phenolics prior to infection reduced disease severity by 20 to 100%, as shown in infection assays in three different hosts. The effect was not species speciic and was proven effective in P. carotovorum, P. carotovorum subsp. basiliense, P. atrosepticum and P. aroidearum. To explore the mechanism underlying these processes, the effect of cinnamic acid (CA) and salicylic acid (SA) on virulence was further investigated in P. aroidearum and P. carotovorum subsp brasiliense. The results clearly indicated that both compounds interfered with the quorum-sensing (QS) machinery of the two species, consequently altering the expression of bacterial virulence factors. While in control treatments, expression of QS-related genes increased over time, exposure of bacteria to nonlethal concentrations of CA or SA inhibited the expression of QS genes, including expI, expR, PC1_1442 (luxR transcriptional regu- lator) and luxS (a component of the AI-2 system). Other virulence genes known to be regulated by the QS system, such as pecS, pel, peh and yheO, were also down-regulated relative to the control. In agreement with the low levels of expression of expI and expR, CA and SA also reduced the level of N-acyl-homoserine lactone (AHL) signal. The effects of CA and SA on AHL signaling was conirmed in compensation assays, in which exogenous application of N-(β-ketocaproyl)-L-homoserine lactone (eAHL) led to the recovery of the reduction in virulence caused by the two phenolic acids. The results support a mechanism by which plant phenolics interfere with Pectobacterium virulence via the QS machinery.

20 Bacterial diseases of potatoes in Chile: A re-emergent sanitary problem

Ivette Acuña, Sandoval, C. and S. Mancilla

Instituto de Investigaciones Agropecuarias, INIA Remehue. Casilla, 24-O, Osorno, Chile.

Potato crop in Chile is positioned in fourth place with an average area in the last 10 years of 51.929 ha, reaching a production and yield average of 1.130.405 tons and 21.9 t / ha (ODEPA, 2014). Con- sidering fresh consumption, potato represents about 50 kg/inhabitant per year. In addition, this crop has a great economic and social importance being produced by nearly 60.000 farmers, 50% of them in southern Chile. Today, the main sanitary problems in potato are late blight, PVY and bacterial diseases. During the 90´s, Pectobacterium atrosepticum (Erwinia carotovora sp atrosep- tica) was described as the main causal agent of soft rot and black leg, with a disease incidence of 40% to 100% as tuber latent infection and producing 20% of yield losses. At that time, the problem was managed using in vitro multiplication in the potato seed production system. However, black leg and soft rot is increasing again in the last 5 years, producing 15% of yield losses mainly due to the use of new irrigation systems, favorable weather conditions and new varieties, among others. During the last potato seasons a new survey was performed to do a collection of plant and tuber isolates. Strains were characterized using speciic PCR primer ECA1f + ECA2r (de Boer et al, 1995) and EXPCCF+EXPCCR (Kang et al, 2003) for P. atrosepticum and P. carorotovorum subsp carotovorum. PCR shows that 13.5% and 64.8% of the strains ampliied with ECA1f + ECA2r or EXPCF+EXPCR primer, respectively. However, 10% of strains did not shows reaction with these primers. Then, these results indicate that new strains are associated with potato causing black leg and soft rot, however, new studies need to be performed to have conclusive results.

21 Characterization of Finnish Pectobacterium carotovorum isolates from potato stems showing blackleg symptoms

Miia Pasanen & Minna Pirhonen

Department of Agricultural sciences, University of Helsinki, Finland.

Pectinolytic enterobacteria isolated from potato stems and tubers in Finland were identiied as Pectobacterium atrosepticum, Pectobacterium wasabiae and Pectobacterium carotovorum subsp carotovorum using phylogenetic analyses and biochemical tests (Pasanen et al. 2013). P. c. subsp. carotovorum strains clustered into two distinct groups in phylogenetic analyses. One of these groups mainly contained strains isolated from potato tubers and the other one mainly included isolates from potato stems. However, none of the P. c. subsp. carotovorum isolates differed statistically from the water-treated control in the amount of blackleg symptoms in a ield assays with vacuum -inoculated seed tubers. Stem isolates were not able to elicit a hypersensitive response in tobacco leaves and they also produced barely a measurable levels of autoinducers in the stationary phase in vitro. Biolog phenotypic tests showed no differences between stem and tuber isolates. In DNA -DNA hybridization analysis stem isolate strain showed around 70% DNA-DNA relatedness with P. c. subsp. carotovorum type strain and in average nucleotide analysis they had less than 95% pairwise similarity value with any P. carotovorum species or subspecies, which is less than the cut off value (>95%) for species. Stem isolates clearly differ from P. c. subsp. carotovorum type strain and do not clearly resemble any other the P. c. subspecies, including P. carotovorum subsp. brasiliense. However, the stem isolates produced the expected amplicon with P. c. subsp. brasiliensis subspecies-speciic primers, suggesting that these kind of strains can be mistakenly identiied as P. carotovorum subsp. brasiliense. Pasanen et al. (2013) Annals of Applied Biology 163(3):403-419.

22 Test methods and ϐield correlations for Dickeya and Pectobacterium spp.

Eisse de Haan and Miriam Kooman

Dutch General Inspection Service for agricultural seeds and seed potatoes (NAK)

Results of the yearly Pectobacterium and Dickeya survey showed a the relative increase of Pec- tobacterium carotovorum subsp. brasiliense as causal agent of blackleg. Therefore, we performed ield experiments to determine the aggressiveness of Pcb as compared to Pectobacterium atrosepticum and Dickeya solani. In addition, different test methods were compared and ield correlations of these methods were determined. Results of these experiments will be presented.

Euphresco III

Assessment of Dickeya and Pectobacterium spp. on vegetables and ornamentals

10-12 November, 2016 University of Helsinki Finland

Programme and abstracts

Programme

Date Time Activity Speaker or Address 13:00 Arrival to Department of Latokartanonkaari 5, THURSDAY Agricultural Sciences 3rd floor, Viikki November 10 Coffee and sandwich campus Room 336 13.15 Welcome and practical Minna Pirhonen and information Jan van der Wolf

PLANT-PATHOGEN INTERACTIONS, ECOLOGY AND MANAGEMENT

13.30 Motility and chemotaxis: Nicole Hugouvieux- phenotypic analysis of Dickeya Cotte-Pattat solani, comparison with Dickeya dadantii 3937 13.50 Competitive fitness: An important Lucy Moleleki survival strategy for Pcb1692 survival? 14.20 Elucidating a mechanism by Iris Yedidia which plant derived small molecules affect quorum sensing related virulence targets of Pectobacterium 14.40-15.00 Coffee break 15.00 Transcriptome profiling of potato Iman Hadizadeh tubers as response to biological control bacterium Serratia plymuthica 15.20 Are waterways important Agata Motyka reservoirs of phytopathogens for the genera Dickeya and Pectobacterium spp.? 15.40 R&D into blackleg caused by Ian Toth Pectobacterium atrosepticum 16.00 Bacteriophages of Soft Rot Robert Czajkowski Enterobacteriaceae – what we know so far? 16.40 Characterization of the pathogens Jianjun Hao that cause blackleg of potato in Maine and their responses to chemical treatments and varieties 17.00 Core and accessory genomes in Frédérique Van the Dickeya genus Gijsegem

20.00 Buffet dinner at Restaurant Hotel Radisson Blu Kitzens Plaza

8.00 Leaving from the hotel FRIDAY 8.17 Buss no 68 to Viikki leaving 8.17 Railway square, November 11 Get off the bus by black gorilla platform 1

DIAGNOSTICS AND SURVEYS 9.00 Improving diagnostics of soft rot Jan van der Wolf Enterobacteriaceae 9.20 Evaluation of real-time assays for Valérie Helias the detection of Pectobacterium and Dickeya 9.40 Classification of potato blackleg Sean Li and soft rot disease in Canada 10.00 Predicting Dickeya by post- Gary Secor harvest seed lot testing: A Real World Study 10.30-11.00 Coffee break 11.00 Pectobacterium and Dickeya Ivette Acuña situation in the potato crop in Chile 11.20 Pectobacterium and Dickeya: use Miriam Kooman of big and small data 11.40 Diagnostic microarray in parallel Yeshitila Degefu detection of bacterial pathogens of potato: hype or opportunity 12.00 Detection of Pectobacterium and Natalie Laughton Dickeya species in irrigation water in South Africa 12.20 One year survey of soft rot Marie-Anne Barny enterobacteria in non host environment Lunch at the student 13.00-14.30 Lunch break and staff restaurant Ladonlukko 14.30 Blackleg in Scotland: Recent Greig Cahill findings 14.50 Dickey solani, isolated Juliana Perminow occurrences in Norway? 15.10 Novel Dickeya species - again? Špela Alič

15.30 Coffee, cake and discussions Jan van der Wolf Staff lunch room 17.00 Finishing the meeting

9.40-9.55 Boat to Suomenlinna Market square by the SATURDAY water November 12 10.00-11.30 Guided tour in Suomenlinna 11.30- Lunch Suomenlinna 13.20-13.35 Boat back to market square Suomenlinna pier 13.35-16.30 Guided tour down town At the market place Helsinki

Abstracts

Motility and chemotaxis: genomic and phenotypic analysis of Dickeya solani, comparison with Dickeya dadantii 3937

Nicole HUGOUVIEUX-COTTE-PATTAT

CNRS UMR5240 Microbiologie Adaptation et Pathogénie, Université de Lyon, Université Claude Bernard Lyon 1, INSA de Lyon, F-69621Villeurbanne Email: [email protected]

Motility and chemotaxis are crucial for the colonization of the host plant and a successful infection. Chemoreceptors (MCPs) are the sensory core of the system; they sense and transduce the signal to the flagellar motor. A bioinformatics analysis predicted 43 complete MCP genes into the D. solani genome. Each gene has an orthologue among the 47 MCP genes of D. dadantii and pairs of orthologues share the same genomic context. An experimental approach was also conducted to identify compounds able to induce chemotaxis in D. solani and dadantii. This phenotypic analysis showed a positive chemotactic response towards several sugars, amino acids or organic molecules, with few differences between strains or species.

Competitive fitness: An important survival strategy for Pcb1692 survival?

MOLELEKI LN1,2* TANUI CK1 AND SHYNTUM D1

1Department of Microbiology and Plant Pathology, 2Forestry and Agriculture Biotechnology Institute University of Pretoria, Lunnon Rd, Pretoria Email: [email protected]

Pectobacterium carotovorum subsp brasiliense (Pcb) is an important emerging soft rot and blackleg pathogen of potato plants globally, for which relatively little is known. This work uses the sequenced genome strain (Pcb1692) to investigate important virulence factors of this emerging pathogen. Towards this end, global genome-wide transcriptome profiling (RNA_Seq) of Pcb cells, during colonisation of potato tubers, was undertaken. From the data generated, it is evident that Pcb1692 utilises various strategies to kill or inhibit competing ‘non-self’ neighbouring bacteria cells residing within potato tubers. This presentation will explore these transcriptome data in more detail and discuss arising hypotheses.

Elucidating a mechanism by which plant derived small molecules affect quorum sensing related virulence targets of Pectobacterium

YEDIDIA I1* AND JOSHI RJ1,2

1The Institute of Plant Science, Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel; 2Department of Agro-ecology and Plant Health, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel Email: [email protected]

The potential of plant derived phenolic molecules to control soft-rot caused by Pectobacterium spp. was investigated. To date, efforts to control the disease were mainly dedicated to improve detection methods and sanitation during storage and agricultural practice, with a limited degree of success. Here we tested the antimicrobial potential of different active phenolics on Pectobacterium, in search for a possible mode of action. Interestingly, biofilm formation and exoenzymes activity were significantly impaired, at compounds concentration that did not affect bacterial cell growth. These observations suggested a mechanism which specifically interferes with bacterial virulence. Since, these major virulence determinants (biofilm and exoenzymes) are controlled by quorum-sensing (QS), we focused on the effect of specific molecules on QS system in pectobacteria. The study revealed an inhibiting effect of the tested compounds on the expression level of central QS system and QS controlled genes, using quantitative real time-PCR. Two reporter strains (CV026 and pSB401) demonstrated a prominent reduction in the level of QS signal molecules N- acyl-homoserine lactone (AHL) accumulation, following exposure to the compounds. Moreover, infection capability was strongly impaired on three different hosts, potato, cabbage and calla-lily; but almost completely recovered upon external application of AHL (exogenous-AHL). To support potential interaction of the plant phenolic compounds with QS targets, drug discovery tools were used (SCHRODINGER®) to reconstruct a computational model of the QS central proteins in Pectobacterium ExpI/ExpR and predict the potential of specific compounds to bind to the active site of these targets.

Transcriptome profiling of potato tubers as response to biological control bacterium Serratia plymuthica

Iman Hadizadeh, Bahram Peivastegan and Minna Pirhonen

Department of Agricultural Sciences, PO BOX 27, 00014 University of Helsinki, Finland Email [email protected]

One of the biggest quality problem in cultivation of potato and vegetables is caused by soft rot bacteria in the genera Pectobacterium and Dickeya. Biocontrol bacterium Serratia plymuthica A30 has a potential to control potato tuber soft rot caused by Dickeya solani at postharvest storage conditions. In this work, potato tubers were treated with A30 and potato transcriptome profiling was performed with RNA-seq to understand the functional response of potato tuber to this biological control bacterium. As a result of the biocontrol treatment, expression of genes involved in photosynthesis, response to stress, hormone signalling pathways and plant defence were induced in the tuber tissue.

Are waterways important reservoirs of phytopathogens for the genera Dickeya and Pectobacterium spp.?

MOTYKA A, SERBAKOWSKA K, BABINSKA W, ZOLEDOWSKA S, SLEDZ W, LOJKOWSKA E

1Department of Biotechnology, Intercollegiate Faculty of Biotechnology University of Gdansk and Medical University of Gdansk, Gdansk, Poland Email: [email protected]

Pectinolytic bacteria from the genera Dickeya and Pectobacterium are causative agents of soft rot and blackleg on potato. Few transmission pathways of these pathogens were reported so far i.a. via the waterways. In this work we examined the survival of Dickeya and Pectobacterium spp. in Polish waters of different pollution status, both rivers and lakes. Additionally, information about the occurrence of pectinolytic bacteria in different waterways and different depths of the water reservoirs will be presented ed. Phenotypic features and the ability to cause potato tissue maceration of the isolated strains was evaluated. The presented data can open debate about the possible contribution of water-derived isolates to economic losses in potato production sector.

R&D INTO BLACKLEG CAUSED BY PECTOBACTERIUM ATROSEPTICUM

I K Toth1, G Cahill2, J G Elphinstone3, S Humphris1, G S Saddler2 and S J Wale4

1James Hutton Institute, Invergowrie, Dundee DD2 5DA 2Science and Advice for Scottish Agriculture (SASA), Roddinglaw Road, Edinburgh, EH12 9FJ 3Food and Environment Research Agency (Fera), Sand Hutton, York, YO41 1LZ 4SRUC Aberdeen Campus, Craibstone Estate, Aberdeen AB21 9YA E-mail: [email protected]

Pectobacterium atrosepticum continues to be the major issue for potato production in Northern Britain, while other species and genera are of concern in mainland Europe. A three year project, funded by AHDB Potatoes and the Scottish Government, has recently been commissioned to identify how and when early field generations become infected by P. atrosepticum, together with the effectiveness of sulphuric acid in haulm destruction. The project has 5 main components:-

• Monitor commercial first generation crops through a 3 year multiplication cycle. • Investigate the movement of P. atrosepticum from infected to healthy plants. • Investigate the routes by which daughter tubers become infected once P. atrosepticum is present in or on a plant. • Identify whether a change in the population of P. atrosepticum strains has occurred in recent years. • Use of modelling to better understand the geographical distribution of infected seed crops. • Compare the effectiveness of sulphuric acid with currently used haulm destruction programmes to determine their relative impact on spread of P. atrosepticum to daughter tubers.

This presentation will summarise the findings from the project.

Bacteriophages of Soft Rot Enterobacteriaceae – what we know so far?

Robert Czajkowski, Anna Smolarska, Zofia Ozymko

Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Abrahama 58, 80-307 Gdansk, Poland e-mail: [email protected]

Soft rot Enterobacteriaceae (SRE: Pectobacterium spp. and Dickeya spp., formerly pectinolytic Erwinia spp.) are ubiquitous necrotrophic bacterial pathogens that infect a large number of different plant species worldwide, including economically important crops. Despite the fact that SRE bacteria have been studied for more than 50 years, little is known about their corresponding predators: lytic and lysogenic (temperate) bacteriophages. Lytic bacteriophages are able to shape the structure of host population and therefore may influence bacterial virulence, spread, persistence in environment and evolution. Up till now, ca. 6000 different individual bacteriophage isolates have been described and visualized by transmission electron microscopy (TEM), of which about 2000 (ca. 30%) target members of the Enterobacteriaceae family. In contrast, the number of characterized bacteriophages infecting the soft rot Enterobacteriaceae (SRE) bacteria (Pectobacterium spp. and Dickeya spp.) is much fewer and less than twenty. Likewise, the first (complete and/or draft) genomes of SRE bacteriophages were published as recently as in 2012. This presentation acknowledges past and present work on lytic (and lysogenic) bacteriophages able to infect Pectobacterium spp. and Dickeya spp. with the major focus on isolation of (novel) lytic bacteriophages from environmental samples and their morphological, proteomic, phenotypic and phylogenetic characterization. As well, the use of next generation sequencing and bioinformatic technologies in analyses of bacteriophage genomes and comparative phage genomics will be discussed.

Characterization of the pathogens that cause blackleg of potato in Maine and their responses to chemical treatments and varieties

Hao JJ1* Marangoni N1 Jiang H1 Song Y1 Ge T1 and Johnson SB2

1School of Food and Agriculture, 2Cooperative Extension University of Maine, Orono, ME 04469, USA *Email [email protected]

Blackleg of potato is a newly emerging disease in the Eastern states of the US. The pathogen population consisted of Dickeya dianthicola, Pectobacterium wasabiae, and P. caratovorum being the major species. D. dianthicola was the predominant species and could be the reason of the outbreak since 2015. The potato variety ‘Sebago’ was the most tolerant to P. carotovorum; AF5312-1 was the most tolerant to P. wasabiae; ‘Yukon Gold’, AF 4172-2, and ‘Shepody’ were the most tolerant to Dickeya spp. In chemical treatments, oregano essential oil and isothiocyanate inhibited both Dickeya spp. and Pectobacterium spp.

Core and accessory genomes in the Dickeya genus

PÉDRON Jacques and VAN GIJSEGEM Frédérique Institute of Ecology and Environmental Sciences – Paris INRA-UPMC-CNRS-IRD-Université Paris Diderot-UPEC Paris, France Email: [email protected]

Multiple sequencing of genomes belonging to a bacterial genus or species allows one to analyse and compare the gene complements of genus or species, their pan- genomes. Such a study reveals species diversity and gene families that may be of special interest, e.g because of their role in bacterial survival/virulence or their ability to discriminate strains. We began such an analysis for the Dickeya genus by comparing 23 genomes available in databases using a SILIX-based clustering of the ORFs sharing at least 80% identity using Blastp scores. This allowed us to define a Dickeya genus core genome including the genes shared by all the analysed bacterial strains as well as core genes shared by different phylogenetic clades in the Dickeya phylogenetic tree. We also identified the genes that are specific to the different Dickeya species. These data will be discussed in terms of species specificity and link to virulence potential.

Improving diagnostics of soft rot Enterobacteriaceae

Jan van der Wolf, Marjon Krijger, Pieter Kastelein, Lia de Haas, Patricia van der Zouwen, Theo van der Lee, Peter Bonants, Elio Schijlen, Jan Bergervoet

1 Wageningen Plant Research P.O. Box 16, 6700 AA Wageningen, the Netherlands Email [email protected]

Testing of plant propagation material for soft rot Enterobacteriaceae (SRE), but also studies on ecology and disease management, require the availability of reliable diagnostic methods. Methods for characterization and testing of plant propagation material currently in use or recently developed will be shortly evaluated. These include methods for 1. Isolation, 2. characterization of bacteria using multilocus sequence analysis, 3. testing for virulence, 4. Enrichment of SRE, 5. detection with TaqMan and LAMP assays, 6. multiplex detection with the (Luminex) xTaq technology, and 7. NGS-analysis in complex substrates.

Evaluation of real-time assays for the detection of Pectobacterium and Dickeya

HELIAS V 1, 2, QUETU-LAURENT A 1, 2, CHAWKI K 1, 2, AND ANDRIVON D 2

1 FN3PT/RD3PT, French Federation of Seed Potato Growers /Research, Development, Promotion of Seed Potatoes, 43-45 rue de Naples, F-75008 Paris, France 2 INRA, UMR1349 IGEPP, F-35653 Le Rheu Cedex, France Email: [email protected]

The French Federation of Seed Potato Growers leads, in collaboration with INRA, a research program on Pectobacterium and Dickeya including assessment and development of methodologies for the detection of bacterial species involved in potato crop damages. The presented work aimed to evaluate the specificity and the sensitivity of several real-time PCRs protocols. The study focused on 7 real-time of the 11 assays initially selected for the Euphresco ring test for the detection of P. atrosepticum, Dickeya spp., D. solani, D. dianthicola, P. brasiliense and P. parmentieri, including Taqman and Sybergreen technology. The test performance study evidenced differences in specificity and sensitivity. Comparisons will be enlarged to other real-time assays in case of the availability of alternative tests for the detection of the same target organisms.

Classification of potato blackleg and soft rot disease in Canada

LI X, NIE J, ARSENAULT, H, XU H, AND De BOER SH.

Canadian Food Inspection Agency, Charlottetown Laboratory Email: [email protected]

Dickeya spp. and Pectobacterium spp. have caused substantial losses globally due to the emergence of new species/subspecies, and the wide spread dispersion of these pathogens which are more virulent and pathogenic in a wide variety of hosts. Particularly in potatoes, Dickeya spp and Pectobacterium spp can cause blackleg and blackleg-like diseases which have become increasingly problematic for seed potato production in many potato growing regions. In this report, we summarized the identification, characterization and classification of potato isolates of these two genera obtained during the last few years. In North America, P. atrosepticum, P. kelmani (a potato isolate of P. wasabiae), and P. carotovorum subsp. brasiliensis were routinely detected during the seed potato indexing, whereas Dickeya spp are relatively rare in seed potato lots. D. solani has not been detected in potatoes in Canada. However, Dickeya solani infecting hyacinth bulbs imported from overseas may be a potential threat to the local potato industry.

Predicting Dickeya by post-harvest seed lot testing: A Real World Study

GARY SECOR1, JOHN NORDGAARD2 and VIVIANA RIVERA1.

1Department of Plant Pathology, North Dakota State University, Fargo, North Dakota, USA 58102, 2Black Gold Farms, Grand Forks, North Dakota, USA 58201

Stand losses due to Dickeya dianthicola is new in the USA. Most infection of seed lots is latent and cannot be detected visually, and seed lots that appear free of Dickeya may exhibit serious stand losses after planting. Post-harvest PCR testing can detect latent seed infection, but there is little data connecting latent Dickeya seed infection with Dickeya in the field. We used PCR to test tuber samples of seed lots destined for commercial planting and monitored stand losses due to Dickeya. There appears to be a strong association between latent Dickeya infection and stand losses in commercial fields. In the USA, it appears that post-harvest seed lot testing can be used to predict stand losses due to Dickeya.

Pectobacterium and Dickeya situation in the potato crop in Chile

ACUÑA, I.1; GUTIÉRREZ, M.2; SANDOVAL, C.1 and S. MANCILLA1

Instituto de Investigaciones Agropecuarias, INIA Chile. Laboratorio Regional Osorno, Servicio Agrícola y Ganadero SAG Chile E-mail: [email protected]

Bacterial diseases in the potato crop have been increasing in the last years, due to various factors, among those the quality of potato seed, the use of irrigation systems and the climate change. These factors have led to an increase in the incidence and severity of black and soft rot caused by P. atrosepticum and P. carorotovorum subsp carotovorum with losses of up to 30% on susceptible cultivars and rejection of seed lots up to 24%. Added to this, Dickeya has been described in table stock potato production in the central part of the country. Due to this situation, it is necessary to develop epidemiology studies, seed diagnostic techniques and integrated management strategies to solve this sanitary problem.

Pectobacterium and Dickeya: use of big and small data

Miriam Kooman and Robert Vreeburg.

Dutch General Inspection Service for agricultural seeds and seed potatoes (NAK)

Since 2012, large scale-post harvest tests have been performed on Dickeya and Pectobacterium in Dutch seed potatoes. Both the field inspection results and data from the post-harvest tests are now available in a data warehouse and can be combined and analysed to find predictive correlations. Initial results from these analyses will be presented, like year-to-year correlations, lab-to-field correlations and (anonymised) variety data. The data can be combined with data from external sources to perform (big) data analyses.

Diagnostic Microarray in Parallel Detection of Bacterial Pathogens of Potato: hype or opportunity

YESHITILA DEGEFU

Natural Resources Institute Finland, Green Technology, Biotechnology and Genetic Resources Paavo Havaksen tie 3, FI-90014 University of Oulu, Finland. [email protected]

Mixed infections with different pathogens are common in plants, which challenges pathogen detections by increasing analysis cost and time. Therefore, methods allowing parallel detection of different pathogens are needed. Potato tubers are infected with many bacterial pathogens whose detection is necessary in seed certification and quality control of ware potato stocks. In this study, a diagnostic microarray previously tested for specificity of probes for detection of eight known major potato bacterial pathogens was evaluated for routine and end user applications. Results of investigations involving tuber samples and samples emulating actual sample (tuber) are presented. While diagnostic microarray should not be considered unfit, it does not quite live up to the hype either. Details will be discussed.

Detection of Pectobacterium and Dickeya species in irrigation water in South Africa

LAUGHTON NH, CHIDAMBA L AND VAN DER WAALS JE

Plant Pathology Programme, Department of Plant and Soil Sciences, Private Bag X20, Hatfield, Pretoria, 0028, South Africa Email: [email protected], [email protected]

Soft rotting Enterobacteriaceae (SRE) such as Pectobacterium and Dickeya spp. are responsible for causing black leg and soft rot diseases of potatoes. These diseases are of major concern to the potato industry throughout the world. Detection and identification of Pectobacterium and Dickeya species in water sources used for irrigation is extremely important for management of the diseases. In South Africa no studies have been done to identify the presence or prevalence of either genus in water systems. Five potato growing regions in South Africa were sampled. Water samples from dams, boreholes, rivers and overhead irrigation systems were surveyed for Pectobacterium and Dickeya species. Identification was done by REP- PCR and Multiplex PCRs, using species specific primers. Initial tests revealed that SRE are detectable in water systems used for irrigation in most regions sampled. Notably SRE were detectable in all overhead irrigation systems sampled. This is an important finding for the South African agricultural industry.

One year survey of soft rot enterobacteria in non host environment

BARNY MA1, VAN GIJSEGEM F1 and BERGE O2

1 Institute of Ecology and Environmental Science-Paris University Pierre and Marie Curie - 4 place Jussieu - 75252 PARIS cedex 05, 2INRA,Plant Pathology Research Unit, 84143 Montfavet, France Email: [email protected]; [email protected]

Soft rot enterobacteria (SRE: Pectobacterium spp. and Dickeya spp.) infect a large number of plant species worldwide, including economically important plants. While the diversity of SRE observed on plant is fairly well described, the presence and diversity of SRE outside the plant context is not known in detail. In this study we sampled SRE outside the plant context. In october 2015, february, may and august 2016 we sampled on 21 sites along the river Durance in the south of France. Nearly 300 strains were isolated and we will present the results of this year survey.

Blackleg in Scotland: Recent findings

GREIG CAHILL AND GERRY SADDLER

Science and Advice for Scottish Agriculture (SASA) Edinburgh, Scotland (UK) Email: [email protected] & [email protected]

Blackleg continues to be the principal cause of downgradings and rejections of seed crops from the Scottish seed potato classification scheme (SPCS). In Scotland, unlike other parts of Europe, Pectobacterium atrosepticum remains the predominant pathogen found. Multi-locus sequence analysis (MLSA) confirms that although P. atrosepticum is a diverse species there is little evidence to suggest that new pathogenic groups have emerged to explain the recent increase in disease levels. Results from MLSA may also indicate that environmental sources are just as important and seed-borne infection. These and other results are helping to shape the SPCS and during the 2016 growing season seed classification scheme rules were tightened, introducing a third inspection for ‘at risk’ crops. Initial results from this trial will be discussed in addition to outlining future plans.

Dickey solani, isolated occurrences in Norway?

J.I.S. Perminow, M.B. Brurberg NIBIO Biotechnology and Plant Health Norway Email: [email protected]

Potato blackleg and soft rot are bacterial diseases of potato. Symptoms include dark discoloration of the stem base and aerial stem rot. Damaged vascular tissue leads to wilting of the affected plants. Soft rotting of tubers also occurs. There are several bacteria that can cause the described symptoms, Pectobacterium sp. and Dickeya sp. In 2005, a new aggressive Dickeya solani was detected in the Netherlands, and in the following years in the majority of potato producing countries in Europe. In Norway, an isolated detection of Dickeya solani took place in 2012 in the course of an official survey. The finding was made in potato plants grown from imported seed potatoes under quarantine conditions, after which plants were destructed. In 2015 Dickeya solani was detected in three Norwegian field trial locations.

Novel Dickeya species - again?

ALIČ Š1,2, VAN GIJSEGEM F3, NAGLIČ T1,4, PÉDRON J3, TUŠEK-ŽNIDARIČ M1, , RAVNIKAR M1,4, DREO T1,4*

1National Institute of Biology, Dept. of Biotechnology and Systems Biology, Slovenia, 2Jožef Stefan International Postgraduate SchoolSlovenia, 3UPMC Univ Paris 06, INRA, IEES Paris (Institute of Ecology and Environmental Sciences), France 4COBIK - Centre of Excellence for Biosensors, Instrumentation and Process Control, Centre for Biotechnology,Slovenia *Corresponding author: [email protected]

Bacteria isolated from diseased tissues of Phalaenopsis orchids from a commercial production site were identified as Dickeya spp.. Comparative genomic study of genomes of the two selected isolates B16 and S1 (Alič et al., 2015) and publicly available Dickeya genomes identified the isolates as a putative new species within genus Dickeya, along with MK 7 and NCCPB 3274 strains. Based on partial sequencing of fliC the isolates are similar to Dickeya isolates from Asia (Suharjo et al., 2014) indicating that the new species may be widespread in nature. Strains of the putative new species show unique carbon assimilation profile and high aggressiveness on chicory leaves. Isolates B16 and S1 were able to systemically colonize potato plants.

st nd 1 and 2 November 2017 SASA, Edinburgh, Scotland, UK

SASA, Edinburgh, Scotland, UK 1st & 2nd November, 2017

08:50 Meet in Hotel foyer for optional tour. (See additional information) 09:30-11:00 Tour of Edinburgh vaults 11:15 Bus back to SASA from Old Town (private bus) 11:30 Pick up from hotel for those who didn’t attend tour 12pm Arrive at SASA for Lunch 12:45-13:00 Introduction – Gerry Saddler

(Chair, Ian Toth)

13:00-13:20 Greig Cahill, SASA, Edinburgh: Blackleg in Scotland - The current picture 13:20-13:40 Yeshitila Degefu, National Resources Institute Finland (Luke): Observation on latent occurrence and co-occurrence of Dickeya and Pectobacterium species in potato tubers. 13:40-14:00 Jacquie van der Waals, University of Pretoria: Detection of soft rotting Enterobacteriaceae in irrigation water used in potato production systems in South Africa. 14:00-14:20 Yves le Hingrat, FN3PT, Paris: The national research program on blackleg disease in France: what are the main outcomes? 14:20-14:40 Rachel Mann, AgriBio, Victoria: Dickeya dianthicola in Australia 14:40-15:00 Gary Secor, NDSU, Fargo: Updates on Detection and Spread of Dickeya in the US. 15:00-15:30 Tea/Coffee

(Chair, Gerry Saddler)

15:30-15:50 Sonia Humphris/Ian Toth, JHI, Dundee: An investigation into the interplay between metabolism and pathogenicity in Pectobacterium through the Entner-Douderoff pathway. 15:50-16:10 Jakub Fikowicz Krosko, University of Gdansk: Fast and reliable screening system to preselect candidate Dickeya solani Tn5 mutants in plant tissue-induced genes. 16:10-16:30 Frederique van Gijsegem, INRA-UPMC, Paris: New insights into the genomic diversity of Dickeya solani and Dickeya dianthicola. 16:30-16:50 Leighton Pritchard, JHI Dundee: Pectobacterium atrosepticum subspecies structure in Scotland. 16:50-17:10 Ewa Łojkowska, University of Gdansk: Pangenome of plant pathogenic bacterium Pectobacterium parmentieri. 17:10-17:30 Discussion and round up 17:30 Bus leaves for Haymarket Hub Hotel 19:30 Dinner at First Coast, Dalry Road (see Additional Information)

TYPE/PSHOP 1

08:00 Pick up from Hotel

(Chair, Ewa Łojkowska)

09:00-09:20 Mirjam Prinz, Bavarian State Research Center for Agriculture, Freising: Development of a multiplex assay for blackleg bacteria directly applied to potato tuber sap. 09:20-09:40 Minna Pirhonen, University of Helsinki: Potato dextrose agar as an easy way to identify Dickeya solani. 09:40-10:00 Jeremy Cigna, SIPRE, France: Rapid method to identify pectinolytic bacteria isolated from blackleg symptoms in potato fields: Development and application of the “gapA” tool.

(Chair, Minna Pirhonen)

10:00-10:20 Jan van der Wolf, Wageningen UR: Suppressiveness of potato against Dickeya solani 10:20-10:40 Amélie Beury, SIPRE, France: Introduction of two biocontrol bacteria into potato plant rhizosphere to prevent latent contamination by pectinolytic bacteria naturally present in soil and blackleg symptom development. 10:40-11:00 Tea/Coffee 11:00-11:20 Ivette Acuña, INIA, Chile: Potato seed treatment as a part of an integrated management of blackleg and soft rot in potato. 11:20-11:40 Brice Dupuis, Agroscope: Occurrence and aggressiveness of Pectobacterium and Dickeya strains in Switzerland. 11:40-12:00 Robert Vreeburg, NAK, Emmeloord: Initial infection of seed potato lots with P. brasiliense. 12:00-12:20 Doretta Boomsma, HZPC Research B.V. Netherlands: Evaluation of the field tolerance of potato cultivars to single and mixed infections of different Dickeya/Pectobacterium species. 12:20-12:40 Alison Blackwell, APS Biocontrol Ltd: Developing Bacteriophage for the Management of Blackleg. 12:40-13:25 Lunch (Chair, Jan van der Wolf)

13:25 – 13:45 Marie-Anne Barny, Institute of Ecology and Environmental Science, Paris: Phenotypic characterization on plant of soft rot enterobacteria isolated outside the plant context. 13:45 – 14:05 Guy Condemine, University of Lyon: Identification of new Dickeya dadantii virulence factors: a Tn-seq approach. 14:05 – 14:25 Vladimir Gorshkov, Kazan Institute of Biochemistry and Biophysics, Russia: Stealth and brute force behaviour of Pectobacterium atrosepticum inside the plant. 14:25 - 14:45 Marta Potrykus, University of Gdansk: The influence of two quorum sensing systems on the phenotypic features of Dickeya solani exhibiting different virulence levels. 14:45-15:05 Iris Yedidia, Volcani Centre, Israel: Interkingdom signalling: interference of plant derived small molecules with bacterial communication and virulence. 15:05-15:25 Patrice de Werra, Bern University of Applied Sciences: Transmission of blackleg symptoms and latent infection from plant to plant under greenhouse conditions. 15:25-16:00 Tea/Coffee and final discussion 16:00 Close 16:30 Bus departs to hotel Taxis to airport

TYPE/PSHOP 2

Greig Cahill, Gerry Saddler Science and Advice for Scottish Agriculture (SASA), Edinburgh, Scotland (UK) Email: [email protected] & [email protected]

Blackleg continues to be the principal cause of downgradings and rejections of seed crops in Scotland. Recent changes to seed certification which introduced a compulsory third inspection under some circumstances will be discussed. In Scotland, unlike other parts of Europe, Pectobacterium atrosepticum remains the predominant pathogen causing infection, and since 2010 annual surveys of tubers and crops have confirmed the absence of Dickeya spp. Results from these surveys will be presented, including the status of Pectobacterium wasabiae and Pectobacterium carotovorum subsp brasiliense present in Scotland. The results of a pilot project testing Pre-Basic crops for latent Pba infection will also be discussed.

Yeshitila Degefu, Natural Resources Institute Finland (Luke), Green Technology Unit, Paavo Havaksen tie 3, P.O.Box 413, FI-90014 University of Oulu, Finland. [email protected]

The Enterobacteriaceae is a large family of Gram-Negative bacteria that includes, in addition to many harmless symbionts, the more familiar human bacterial pathogens and the bacteria in the genera Dickeya and Pectobacterium which cause blackleg and soft rot in potato. Dickeya solani, Dickeya dianthicola, Pectobacterium atrosepticum, Pectobacterium carotovorum subsp., Pectobacterium brasiliense and Pectobacterium wasabiae are the currently known enterobacteriacae causing blackleg and soft rot in potato in Europe and globally. The Bacteria latently present in the tuber are the main sources of infection and it is often observed that seed lots and the potato plant could be contaminated by one or more of these species. Since 2005 the lab at the now Natural Resources Institute Finland (Luke) has been providing seed tuber testing (diagnostic) services to potato growers and seed potato production and distribution companies in Finland besides the blackleg and soft rot research. The test results have been carefully documented. This presentation highlights the results of the thirteen years of seed testing and summarizes the occurrence and co-occurrence of the bacterial species during latent survival in the tubers. Which species appear happy together and which ones, according to the statistics of occurrence, cohabit less frequently or even did not appear to share the tuber niche? The presentation is based only on observation of analysis test results of nearly 1500 samples over a thirteen year period and it is simply preliminary data which requires further investigation in vitro and in planta. At this time where researchers are scratching the surfaces for possible control measures of blackleg and soft rot in potato, it might be useful to get some insight about the meaning of the diversity and interactions among these threatening bacterial pathogens of potato. Could it be possible that Dickeya and Pectobacterium species nourish their own controls?

van der Waals, J.E., Laughton, N., Chidamba, L. & Korsten, L. Department of Plant and Soil Sciences, University of Pretoria, Private Bag X20, Hatfield, 0028

In South Africa, up to 85% of potatoes are produced under irrigation; hence the need to evaluate the prevalence of Pectobacterium and Dickeya species in agricultural water sources for more effective disease management strategies. A total of 104 water samples were collected from 50 sampling points including dams (20), boreholes (3), rivers (6) and overhead irrigation systems (21) in seven potato growing regions of South Africa. Crystal violet pectate selective culturing of SREs from enriched water samples, isolation and PCR identification of the various species, detected the pathogens in water systems used for irrigation in all of the sampled regions except one. Species which are known to cause diseases in potatoes including Dickeya spp., Pectobacterium carotovorum subsp. brasiliense, Pectobacterium carotovorum subsp.

TYPE/PSHOP 3 carotovorum, Pectobacterium atrosepticum and Pectobacterium parmentieri were identified from the water sources sampled. Preliminary pathogenicity evaluation of the isolates showed that some can cause soft rot of tuber slices. This is an important finding for the South African potato industry to profile the disease-causing potential of agricultural irrigation water.

Hélias Valérie1,2, Chawki Khaoula1,2, Quêtu-Laurent Angélique1,2, Andrivon Didier2, Le Hingrat Yves1

1FN3PT/RD3PT, French Federation of Seed Potato Growers, 43-45 rue de Naples, 75008 Paris, France 2INRA, UMR1349 IGEPP, 35653 Le Rheu Cedex, France

Since 2003 the FN3PT has been leading a research program on Pectobacterium and Dickeya in collaboration with INRA. The four themes of the program are diversity, detection, epidemiology and control of the disease.

Field surveys, together with diversity studies of the resulting strain collections, showed that blackleg symptoms are associated with a much larger range of species than the one P. atrosepticum which was historically almost exclusively held responsible for these symptoms in Europe. A collaborative project with the CFPB-CIRM was developed for analyzing more deeply the diversity and the phylogeny of Pectobacterium using MLSA. Pectinolytic bacteria identification was also performed using gapA (presentation of Cigna et al.).

Consequently, the availability of high performance, fast and reliable detection tools for all members of this genus is of utmost importance for both seed certification and disease control. Recent evaluations of the performance (sensitivity, specificity and detection thresholds) of several published real-time assays contribute to that objective.

Molecular detection methods were used to assess bacterial contamination on tubers during a French-Swiss concerted action (FN3PT-Bern Univ. Plant Sciences). One of the objectives of the project was to find out if evaluations of latent infections improve the prediction of subsequent disease development risk.

In parallel with the development of biocontrol strategies, (see Munier et al. presentation) the use of genetic resistance constitutes an interesting prospect in the control of blackleg disease, which currently relies only on prophylactic measures. Over the last decade, FN3PT has observed changes in the bacterial complex responsible for soft-rot and blackleg. These changes have led to the evaluation of 24 clones (CRBBraCySol) which were identified by INRA in three Solanum species in the late 1990’s. Previous studies demonstrated that these clones have a resistance to P. atrosepticum but no information existed up to now on resistance levels of genetic resources for these newly identified bacterial groups. The recent outcomes of the different studies will be presented. "

Rachel Mann1,2, Monica Kehoe3, Elisse Nogarotto1,2, Dominie Wright3, Brenda Coutts3, Brendan Rodoni1,2

1AgriBio, Agriculture Victoria Research, Bundoora, VIC, Australia 2Plant Biosecurity Cooperative Research Centre, Canberra, ACT, Australia 3Department of Primary Industries and Regional Development, South Perth, Western Australia

In May 2017, uncharacteristic blackleg symptoms were observed by an inspector in potato crops in Western Australia. The causative organism was identified as Dickeya dianthicola at the Department of Primary Industries and Regional Development and confirmed at secondary laboratories in New South Wales and the Northern Territory, Australia. This is the first time D. dianthicola has been detected in Australia. An immediate incident response was put in place by the Western Australia government with surveillance and tracing activities implemented. D. dianthicola was detected in seed potatoes, dormant dahlia tubers and freesia bulbs and tracing of these hosts occurred to and from infected properties. This presentation will outline the outbreak, with a focus on the diagnostics.

TYPE/PSHOP 4

Gary Secor, Viviana Rivera-Varas and Blake Greiner. Department of Plant Pathology, North Dakota State University, Fargo, ND 58102 USA

This presentation will provide updates on detection and spread of Dickeya in potatoes. Dickeya dianthicola is an important component of the potato soft rot complex in the US causing serious field stand loss. Warmer temperatures are necessary for expression of Dickeya symptoms, but infection is usually symptomless in seed potatoes produced in cool climate areas. Consequently, it is necessary to test seed tubers for latent Dickeya infection using a PCR test. Dickeya was detected in the stem end, the tuber shoulder, peel strip, and sprout, but the peel strip has the highest frequency of detection compared with the other tissues. Lenticels may play an important role as source of infection in seed tubers, and PCR testing both stem end core and peel samples may be necessary to maximize latent Dickeya detection in seed lots. Field trials failed to show spread of Dickeya during seed handling and cutting operations. An irrigated field trial was conducted to detect the spread of Dickeya from infected seed to adjacent plants and tubers during the growing season. Low amounts of seed decay and blackleg were observed in adjacent plants and Dickeya was detected in only a few of the tuber samples by post-harvest PCR testing.

Huan Wang1,2, Shuo Du1, Yujie,Wang1, Jiaqin Fan1 Sonia Humphris2 and Ian Toth2

1Nanjing Agricultural University, Nanjing, 210095, China 2The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK

The Entner-Douderoff (ED) pathway is one of the alternative glycolytic pathways in prokaryotic glucose metabolism. Our previous work has shown that eda (encoding 2-keto-3-deoxy-gluconate-6P aldolase), which is a key enzyme in the ED pathway and the breakdown of pectic substances in the plant, is required for full virulence in P. carotovorum subsp. carotovorum (Pcc) strain PccS1. Comparison of genomes showed PccS1 possesses an incomplete ED pathway (missing gene edd, which encodes enzyme 6-phosphogluconate dehydratase), while P. atrosepticum (Pba) has a complete pathway. A deletion mutant of eda in Pba showed attenuated virulence and was unable to effectively degrade pectin, whereas a deletion mutant of edd had no effect on virulence or pectinase activity. Our results show that eda plays an important role in the virulence of Pba but this appears to be through the degradation of pectin rather than through the ED pathway

Jakub Fikowicz-Krośko1, Robert Czajkowski1 1Intercollegiate Faculty of Biotechnology University of Gdansk and Medical University of Gdansk

In this study we presented a simple and fast screening method to preselect candidate Dickeya solani Tn5 mutants which carry random transposon insertions. The Tn5 mutants are generated using mini-Tn5 transposon carrying inducible promoterless gusA reporter gene (β-glucuronidase) and are screened for the β-glucuronidase positive phenotypes under non-inductive conditions. The bacterial mutants negative in the first screen are then screened for β-glucuronidase phenotypes in the presence of plant tissues. In the study we have used stems, leaves and roots of Solanum tuberosum and Solanum dulcamara. The mutants which are positive in the screen with plant tissues are selected for sequencing of the Tn5 insertion sites directly from bacterial genome. The described method is faster and cost-effective in comparison to standard IVET or expression microarrays and RNAseq. It allows to generate a number of ready-to-use Tn5 mutants revealing up-regulation by plant tissue which can be later selected for further studies.

TYPE/PSHOP 5

Jacques Pédron1, Géraldine Taghouty2, Perrine Portier2 and Frédérique Van Gijsegem1 1iEES Paris (Institute of Ecology and Environmental Sciences), INRA-UPMC Université Paris 06, UMR 1392, F75005, Paris, France 2CIRM-CFBP, INRA – IRHS, F-49071 Beaucouzé, France

D. solani and D. dianthicola were mainly isolated from potato and various ornamentals with some D. dianthicola occurrences also in chicory and tomato. A recent reclassification of all former Erwinia chrysanthemi strains present in the CFBP collection into the different Dickeya species allowed the identification of a new D. solani strain that was isolated from tomato in Guadeloupe. On the other, differences in aggressiveness on potato were reported for various isolates in both species. From the genomic point of view, D. solani was shown to be mainly clonal while D. dianthicola appeared to be more diverse from MLSA analyses. We undertook comparative genomic analyses to unravel the possible links between genomic traits and aggressiveness and/or host of isolation. Both species share more than 3,000 genes including most of the genes that were shown to be involved in virulence in the D. dadantii 3937 model strain. Intraspecific diversities will be discussed.

Leighton Pritchard, Sonia Humphris, Ian K Toth James Hutton Institute (Dundee) Errol Road, Invergowrie, Perth and Kinross, Scotland, DD2 5DA

We obtained complete genome sequences for ≈50 P. atrosepticum (Pba) isolates that were either previously sampled by SASA as part of a long-term study in Scotland, or were derived from a field trial at the James Hutton Institute. The sequenced SASA isolates represent a restricted geographical area, and were isolated in the period 2009-2015. Comparative genomic analyses of the assembled, annotated isolates were able to distinguish between phenotypically-similar Pba and non-Pba isolates, and indicated considerable genomic variation within Pba. The observed genomic variation was divisible into four clearly separated subspecies groups, divisible by ANI, SNP trees and core/accessory gene content. No clear geographic pattern in subspecies incidence across Scotland was seen, and the genomic variation of these Scottish isolates was comparable to the genomic variation of isolates obtained from Australasia, the Americas, Asia, and Europe.

Ewa Łojkowska1, Sabina Zoledowska1, Agata Motyka1, Wojciech Sledz1 and Alessio Mengoni2

1Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, 58 Abrahama Street, 80 307, Gdansk, Poland 2Department of Biology, University of Florence, via Madonna del Piano 6, 50019 Sesto Fiorentino, Florence, Italy

Pectobacterium parmentieri and Dickeya solani are newly established species within Pectobacteriaceae family. Interestingly, we observe significant phenotypic differences in terms of virulence factors production and ability to macerate potato tissue not only among genomically heterogenous strains of P. parmentieri but also among genomically homogenous D. solani strains. In order to elucidate the source of these differences, we sequenced several P. parmentieri. A pangenome study was performed on 8 genomes of P. parmentieri. The pangenome structure of the species P. parmentiri was compared with those of D. solani species. P. parmentieri pangenome includes 3788 core genes, 1201 accessory genes, and 1625 unique genes. Analysis of D. solani pangenome indicate that number of D. solani core genes is very similar and contain 3756 core genes, however, the accessory and the unique genome is much smaller and include 430 and 130, respectively. We were able to determine the presence of all genes encoding well-known virulence factors in core genomes of both species.

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Mirjam Prinz, A. Kellermann, G. Bauch Bayerische Landesanstalt für Landwirtschaft IPZ6a Am Gereuth 8, 85354 Freising

Testing for blackleg bacteria on seed potatoes is economically very important due to massive crop loss in the field and potentially high influence of latent infection on exported tubers.

Current methods to detect different species of bacteria are conventional PCR and quantitative real-time PCR that are applied to DNA extracts of potato tuber sap. However, extraction of DNA is time consuming and costly and thus impractical to use for high throughput screening of seed potatoes.

Therefore, we develop a multiplex real-time qPCR assay that is applied directly on diluted potato tuber sap (DiRT-qPCR). In my talk, I will present the latest status of the developed method including the optimization of the multiplex reaction and treatment of potato tuber sap.

References

Stammler et al., DiRT-qPCR for molecular biological detection of RNA encoded viruses. Acta Horticulturae in press. Czajkowski et al. 2011. Control of blackleg and tuber soft rot of potato caused by Pectobacterium and Dickeya species: a review. Plant pathology 60, 6, 999-1013."

Minna Pirhonen, University of Helsinki

Isolation and identification of bacteria from diseased plant tissues is a time consuming task. When Dickeya solani grows as pure culture on potato dextrose agar (PDA), it grows as distinctive wrinkled and brownish red colonies, enabling fast and efficient identification. Streaking bacterial pure cultures on PDA reveals if the isolate belongs to D. solani species. Furthermore, spreading bacteria directly from diseased tissue on PDA plates may work as a rapid way to isolate and identify D. solani directly from the plant tissue, at least when high concentration of D. solani cells are present in the plant tissue. Not all PDA work equally well, the best is the PDA from Biokar.

Cigna Jérémy1-2, Dewaegeneire Pauline1, Beury-Cirou Amélie1, Gobert Virginie1 and Faure Denis2

1Seeds Innovation Protection Research Environment (SIPRE), rue des champs Potez, 62217 Achicourt, FRANCE 2CNRS-I2BC, ANR-15-CE21-0003, 1 avenue de la terrasse, 91198 Gif-sur-Yvette, FRANCE

SIPRE (Seeds Innovation Protection Research Environment) is mandated by the CNPPT (Comité Nord Plants de Pomme de Terre, a seed potato growers association located in North of France) to bring concrete solutions to seed potato growers problems. SIPRE develops biocontrol strategies against Pectobacterium and Dickeya in collaboration with the FN3PT/RD3PT (French Federation of Seed Potato Growers) with the national research program on blackleg disease in France (cf. presentation of Hélias et al.) and with the CNRS-I2BC (cf. presentation of Munier et al.).

In order to test our biocontrol strategies on current pathogens, we isolated near to 700 pathogenic strains from blackleg symptoms in potato fields over the last 5 years.

To ensure the characterization of all pectinolytic strains isolated, we have developed a rapid and accurate molecular method focused on gapA gene. Diversity and phylogeny of Pectobacterium was also performed using MLSA (cf. presentation of Hélias et al.).

TYPE/PSHOP 7 Firstly, an alignment of gapA genes retrieved from genomes publicly available was performed and specific signature-nucleotides were searched. A phylogenic tree has confirmed the identity of each species of Pectobacterium and Dickeya, and validated the approach.

Then, we developed a single PCR-sequencing “gapA” method in order to characterize quickly the 700 soft rot pectinolytic bacteria isolated in blackleg symptoms of potato fields. Results will be presented. Finally, we will test the biocontrol activity of protection strains previously studied on a panel of contemporary pathogens identified with gapA phylogeny.

Jan van der Wolf, Claudia Coipan, Pieter Kastelein, Marjon Krijger, Jolanda Tom, Natasja Riksen, Els Nijhuis, Sven Warris en Jenny Fokkema*

Wageningen UR, Postbus 16, 6700 AA Wageningen, T 0317-480598, E [email protected] *NAO, Van Stolkweg 31, 2585 JN Den Haag

In two years field experiments, potato seed lots of two different cultivars (ca. 20 per cultivar per year), originating from different locations in the Netherlands were planted in the same field in randomized blocks. The natural background of blackleg causing bacteria in the selected seed lots was low. Prior to planting, seed lots were vacuum-infiltrated with Dickeya solani (106 cfu/ml). Large differences in blackleg incidences were found between seed lots of the same cultivar within a year, which can only be explained by differences in suppressiveness of the seed lots against D. solani, as the genetic background of the seed lots was similar, lots were planted in the same field and were infected with the same densities of the pathogen. To address the question which factor determines suppressiveness, seed lots were analysed for the mineral composition, dry weight and the bacterial microbiome. A weak relation was found between suppressiveness and some physico-chemical properties. We also found a relation between suppressiveness and the presence of specific bacterial taxons. To study the potential involvement, bacteria belonging to these taxons were isolated from seed lots, characterized and analysed for (in vitro) antagonism against soft rot Pectobacteriaceae.

The research was financed by the Ministry of Economic Affairs and by Dutch potato breeding companies, under the coordination of the Dutch Potato Organization (NAO)

Euphrasie Munier1, Pauline Dewaegeneire1, Jérémy Cigna1, Julie Bartier1, Valérie Hélias2, Denis Faure3 and Amélie Beury1

1 SIPRE, Seeds Innovation Protection Research Environment, Rue des champs Potez, 62217 Achicourt 2FN3PT/RD3PT, UMR 1349 IGEPP INRA, Agrocampus Ouest Rennes, Université Rennes, 35653 Le Rheu, France 3CNRS, Institut de Biologie Intégrative de la Cellule UMR 9198 CEA, Université Paris Sud USC 1358 INRA, CNRS, Université Paris Sud, CEA, 1 Avenue de la terrasse, 91 198 Gif-sur-Yvette, France

Two combined biocontrol strategies for potato plants were developed in order to control and maintain at a low level soil-borne pectinolytic bacteria naturally present in the soil microbiota and to minimize tuber infections. These strategies rely on the direct application in-furrow of two bacterial biocontrol agents; including Pseudomonas putida A14H7, exhibiting antibiosis activity (Raoul des Essarts et al, 2016), and Rhodococcus erythropolis R138 (Cirou et al, 2007) exhibiting a quorum-quenching activity. R. erythropolis R138 is able to assimilate quorum-sensing signal molecules such as N-acyl homoserine lactones involved in virulence regulation in pectinolytic bacteria. The effect of this combined treatment on the acquisition of soil-borne pectinolytic bacteria by tubers and blackleg symptoms expression on plants was evaluated. The two biocontrol agents were monitored over time in the soil rhizosphere during the growing period to

TYPE/PSHOP 8 ensure that they were maintained. The field trials were conducted over three successive field generations. The results showed either a non-acquisition of pectinolytic bacteria in tubers or a decrease of blackleg symptoms in potato plants grown from treated soil. Our data suggest that this biocontrol strategy could provide significant protection of potato plants against pectinolytic bacteria in field conditions.

Raoul des Essarts et al, 2016 AEM, 82(1):268-278. doi:10.1128/AEM.02525-15. Cirou et al, 2007 Environ. Microbiol. 9:1511-1522.

Acuña, I1.; Sandoval, C1.; Gutiérrez, M2.; Bermúdez1, A.; Araya1, M. and S. Mancilla1

1Instituto de Investigaciones Agropecuarias, INIA Chile. 2Laboratorio Regional Osorno, Servicio Agrícola y Ganadero SAG Chile E-mail: [email protected]

Seed decay, soft rot and blackleg are considered important diseases in the potato production system in Chile. A survey done by INIA shows that 13.5% and 64.8% of the strains causing the disease are P. atrosepticum and P. c. subsp. carotovorum respectively. Additionally, in the last years Dickeya dadantii also has been described in potatoes. Due to the importance of this problem, the Chilean Innovation Agency (FIA) has financed a new research grant. One of the objectives of this project is to develop an integrated management strategy for potato bacterial diseases, based on a real time PCR assay as a seed rot potential to determine the sanitary risk and preventive measures. During the season 2016-17 a storage experiment was performed to evaluate the efficacy of seed potato treatment to manage seed rot potential and latent infection. Results will be discussed.

B. Dupuis1, Ch. Sagnier1, I. Kellenberger1, S. Schaerer1 1Agroscope, Nyon, Switzerland

Summary: In Switzerland, blackleg is the primary ground for rejection of potato seed lots after field inspection. A new survey of Dickeya and Pectobacterium strains was carried out in Switzerland in 2017. Samples of plants with blackleg symptoms were collected in the post-harvest grow-out fields. In total, 62 plots were sampled, corresponding to 62 seed potato fields harvested in 2016. The samples were analyzed by PCR to identify the pathogens responsible for the infection. In addition, a greenhouse trial was undertaken to assess the aggressiveness of Pectobacterium carotovorum subsp carotovorum, Pectobacterium carotovorum subsp brasiliense, Pectobacterium wasabiae, Pectobacterium atrosepticum, Dickeya dianthicola and Dickeya solani. The aggressiveness of two isolates per (sub)specie was tested. Twenty tubers of cv. Désirée were inoculated by soaking in a bacterial suspensions of each isolate (106 cfu/ml). The tubers were then planted in pots containing 5 L of soil and maintained in a greenhouse at 25°C and 70% of relative humidity. The expression of the symptoms was followed for 35 days after emergence, and the progeny tubers were weighted after harvest. The survey revealed that Pectobacterium carotovorum subsp brasiliense was the most prevalent bacteria, with 51.6% of occurrence in the plots, followed by Pectobacterium carotovorum subsp carotovorum and Pectobacterium wasabiae with both 35.5% of occurrence, by Dickeya dianthicola with 11.3% of occurrence, and finally by Dickeya solani with 4.8% of occurrence. Pectobacterium atrosepticum was never detected. Mixed infections were found in 27.4% of the plots, with 17.7% of plots infected by two distinct (sub)species and 9.7% infected by three distinct (sub)species. The greenhouse trial revealed that Dickeya solani was more aggressive than the other species and also that no symptoms were expressed by the plants inoculated with Pectobacterium carotovorum subsp carotovorum. All bacteria species affected the yield, which was most reduced after inoculation with Dickeya solani.

Acknowledgments: The authors want to thank Peter Frei, Werner Wild, Maud Tallant, Gaël Droz and Loris Socchi for their technical support.

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Robert Vreeburg, NAK (Dutch general inspection service), Randweg 14, 8304 AS Emmeloord

The increase in the fraction of samples of Dutch seed potato that is latently infected with P. brasiliense with advancing generation, can be modelled by assuming a yearly chance of new latent infections (P) and a chance for an infection to disappear (Q). Fitting this model on the data of P. brasiliense gave estimates for P and Q being ~30% and ~10%.

We try to find the infection route that can explain the large infection chance of P. brasiliense. Preliminary data indicates that (aerosols from) surface water is not a likely source. The methodology for detecting P. brasiliense in insects proved to be very insensitive, not allowing to collect quantitative data. Several bulks of insects tested positive by PCR for P. brasiliense. Work is in progress to isolate the bacteria and confirm their identity.

Doretta Boomsma: HZPC Research B.V. Roptawei 4, 9123 JB Metslawier, The Netherlands

In 2015 and 2016 field trials were carried out in the Netherlands to evaluate the field tolerance of about 40 potato varieties to single and mixed infections of 3 different Dickeya/Pectobacterium species. Per variety minitubers were vacuum infiltrated with only water (control), Dickeya solani (Ds), Pectobacterium wasabiae (Pw), P. carotovorum subsp. brasiliense (Pcb), and mixtures of Ds+Pw, Ds+Pcb, Pw+Pcb, so 7 treatments per variety. Per variety and per treatment 12 tubers were planted per plot in 4 replications. All plots were assessed for plant emergence and symptom development in time. After harvest the yield (kg) and the number of tubers/plot were determined. All daughter tubers were divided in 8 equal samples and, after enrichment, tested the presence of Ds, Pw, Pcb and Pa by Taqman PCR. It was shown that disease symptom expression has random patterns that are mostly correlated to the factors year and lot/variety but not to treatment

Alison Blackwell, APS Biocontrol Ltd. Prospect Business Centre, Gemini Crescent, Dundee Technology Park, Dundee DD2 1TY

Blackleg is a major cause of seed potato downgrading and rejections in northern Europe, together with production losses from associated tuber soft rot. Blackleg inoculum (Pectobacterium & Dickeya) is transferred and multiplied between successive field generations and problems in seed multiplication create issues for the whole of the supply chain, with up to 30% of ware crops affected from planting infected seed stocks and post-harvest rots affecting the processing, fresh sales, wholesale and pre-packing industries. The challenge addressed in this study is to develop an innovative, sustainable bactericide treatment applicable across the industry, to both reduce seed tuber infection levels and protect against wastage from tuber soft rots downstream. Treatments under development are based on formulations of naturally- occurring, highly-specific and environmentally-acceptable agents (bacteriophage); data will be presented to support their incorporation into a novel biological-control agent, with the potential to have a significant impact on the UK industry, as well as to provide a marketing advantage to seed-potato exports over major competitors.

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Bertrand C1, Munier E2, Lecomte A1, Beury A2, Berge O3 and Barny Ma1

1Institute of Ecology and Environmental Science-Paris University Pierre and Marie Curie - 4 place Jussieu - 75252 PARIS cedex 05 2SIPRE, Rue des champs Potez, 62217 Achicourt 3INRA,Plant Pathology Research Unit, 84143 Montfavet, France

We previously isolated a large panel of Soft Rot Enterobacteria (SRE) outside of the plant context. A subset of these isolates was tested on a plant panel to assess their potential aggressiveness on plants and compared with type strains isolated from various host plants. The panel of plant tested was chosen among the plants where both Dickeya and Pectobacterium were isolated according to the literature (Ma et al, 2007). Furthermore, for potato, both tubers and plants were tested while two developmental stages were tested for cantaloupe. The inoculation procedures and results obtained will be presented.

Kevin Royet1, Nicolas Parisot2, Agnès Rodrigue1, Erwan Gueguen1 and Guy Condemine1

1Université Lyon 1, INSA de Lyon, CNRS UMR5240 MAP, Villeurbanne, France 2INSA de Lyon, INRA, UMR203 BF2I, Villeurbanne, France

Identification of virulence factor genes by testing individual mutants on plants is a very time and labour consuming method. Development of high throughput sequencing methods allows the test of a collection of hundreds of thousands transposon mutants in a single experiment by the Tn-seq approach. This method allows the detection of mutants with reduced but also increased fitness in the plant. We used this method to identify new virulence factors of Dickeya dadantii on chicory. Virulence factors identified differed from those previously known since diffusible ones (secreted enzymes, siderophores or metabolites) could not be detected by this screen. In addition to genes encoding proteins of unknown function that could be new virulence factors, other genes could be assigned to known biological functions: metabolism, regulation, motility and resistance to stress. This method could be easily implemented to other plant pathogenic bacteria and other plants.

Vladimir Gorshkov1,2, Rim Gubaev1, Amina Daminova1, Olga Petrova1, Marina Ageeva1, Natalia Gogoleva1,2, Olga Parfirova1,2, Bakhtiyar Islamov1, Polina Mikshina1, Yuri Gogolev1,2

1Kazan Institute of Biochemistry and Biophysics, Kazan Science Centre, Russian Academy of Sciences, Kazan, Russia 2Kazan Federal University, Kazan, Russia

*e-mail: [email protected], [email protected] *Tel: +7 (843) 292-72-22

Pectobacteria known to utilize brute force for plant colonization are also considered to behave as stealth pathogens. We have shown that stealth behaviour of pectobacteria is associated with colonization of the primary xylem vessels where bacteria form specific multicellular structures – bacterial emboli. These structures represent products of plant-microbe integration, since their formation and maturation requires the action and the metabolites of both macro- and microorganisms. In a form of bacterial emboli, pectobacteria implement symptomless systemic colonization of the plant. The NGS analysis have revealed the features that distinguish stealth and brute force behaviour of pectobacteria in planta and allowed identification of a switcher (coronafacic acid, cfa) that determined a transition from ”amenable” to “devastating” mode of action. cfa mutant was able to systemically colonize xylem vessels but was impaired in colonizing core parenchyma and causing soft rots.

The study was supported by RSF (No 15-14-10022).

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Marta Potrykus1, Nicole Hugouvieux-Cotte-Pattat2 and Ewa Łojkowska

1Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Abrahama 58, 80-307 Gdansk, Poland 2UMR5240 Microbiologie Adaptation et Pathogénie, Univ Lyon, CNRS, Univ Claude Bernard Lyon 1, INSA Lyon, F-69621 Villeurbanne, France

Dickeya solani strains of distinct origin show genetic homogeneity but significantly differ in their virulence. Dickeya species possess two quorum sensing (QS) mechanisms: the classic one based on N-acyl- homoserine lactone (AHL) signals and a specific one depending on the production and perception of an unknown molecule (VFM). To study the influence of these QS, mutants were constructed by inactivating genes coding for each system. Double mutants were obtained by simultaneous inactivation of genes coding for both QS. The inactivation of both QS in D. solani did not reveal any additive effect on the tested features. The inactivation of vfm genes has generally preponderant effect on that of the exp genes. Thus, VFM- and AHL-QS do not work in synergy to modulate the production of diverse virulence factors and the ability to macerate plant tissue.

Acknowledgements

The work was supported by Polish National Science Center with the project UMO-2014/14/M/NZ8/00501 and UMO-2014/13/N/NZ9/01081.

Iris Yedidia Plant Science Institute, Agricultural Research Organization, Volcani Center, Bet Dagan, Israel

Plants produce diverse array of low molecular-mass compounds, with more than 8,000 phenolics. Here, we provide an example for interference of plant small molecules (phenolics) with bacterial virulence, via inhibition of the quorum sensing (QS) machinery of the soft rot bacterium P. carotovorum. Interestingly, biofilm formation and exoenzymes activity were significantly impaired, at compound concentrations that did not affect bacterial cell growth or membrane integrity. Since biofilm production and exoenzymes are virulence determinants known to be under the strict control of QS, the effect of specific molecules on QS was studied. Common volatile and soluble phenolic compounds were tested for their influence on the expression of central QS system and QS controlled genes. The N-acyl-homoserine lactone (AHL) reporter strains (CV026 and pSB401) demonstrated a prominent reduction in the level of QS signal molecules accumulation, following exposure to the compounds. Moreover, infection capabilities were strongly impaired on potato, cabbage and calla-lily; but fully recovered upon external application of AHL. Pectobacterium central QS proteins ExpI/ExpR were used as targets for salycilic acid and carvacrol (plant defense molecules) using SCHRODINGER® molecular docking suit, to show possible interactions. Finally, by using isothermal calorimetry (ITC), SA and CAR were directly bound to cloned and purified ExpI, by this, experimentally supporting the computational docking results. True binding of plant derived phenolics to bacterial QS synthase protein as a target was demonstrated.

Patrice de Werra, Nicolas Linder, Alan Storelli and Andreas Keiser School of Agricultural, Forest and Food Sciences (HAFL), Bern University of Applied Sciences, Switzerland

In Switzerland, Pectobacterium carotovorum subsp. brasiliense (Pcbr) is widely found as latent infection in imported and national seed lots but does not express systematically visible blackleg symptoms in the field. In contrast, the less frequent Dickeya sp. (Dsp) seems to be, once expressed in the field, more aggressive. Based on these observations, we developed an artificial greenhouse assay to study the transmission of

TYPE/PSHOP 12 blackleg (symptoms and latent infection) from plant to plant on a plain with a downward slope of 8%. Three seed potatoes of two different cultivars were inoculated with either Dsp or Pcbr and planted on the upper part of the plain. Twelve non-inoculated potatoes were planted downstream of inoculated tubers. A drip irrigation system provided water at the top of the plain, allowing the water to flow downwards to the non-inoculated plants. During vegetation, the potato plants were checked for blackleg symptoms. After tuber formation, haulm killing was performed and all daughter tubers harvested and analysed for latent infection. The results of two distinct assays are presented here.

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Both the Airlink 100 bus and the tram from the airport pass the hotel. However, there are currently road works closing Haymarket Terrace eastbound (ie from the airport), with the bus turning left and stopping on Magdala Crescent instead and rejoining Clifton Terrace via Grosvenor Street. You can get off before the hotel at Magdala Crescent or after at West Maitland Street. - see map overleaf.

This express bus service runs from the airport to Waverley Bridge (near Princes Street and the main rail and bus stations).

Buy tickets in advance online, from Visitscotland by UK Arrivals, at the bus stop, or from the driver

 Frequency: Every 10 minutes.  Fare: Single £4.50, return £7.50

Tram fares between the Airport and City centre are:

• Adult single £5.50 • Adult return £8.50

Buy your ticket before you board. Tickets can be purchased from ticket vending machines at the airport tram station. Ticket vending machines accept most debit and credit cards (£3 minimum spend) as well as 5p, 10p, 20p, 50p, £1 and £2 coins. No change is given. Edinburgh Trams recommends purchasing return tickets to make your journeys simpler. Return tickets are open ended meaning the return portion can be used when needed.

Taxis are usually plentiful and are located on the ground floor of the multi-storey car park opposite the main terminal building – see map for location of tram & taxi rank stop.

For further information go to http://www.edinburghairport.com/transport-links

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TYPE/PSHOP 15

If you would like to go on the walking tour, Greig will meet you in the Haymarket Hub Hotel foyer at 8:50am, and we will get a bus to Edinburgh’s Old Town.

(LRT Bus No. 31. Cost £1.60 for single journey, no change given on bus. Greig will bring coins in case people need change)

The true history of the Blair Street Underground Vaults is terrifying enough – so abandon thoughts of manufactured frights and make-believe. The skill is in the storytelling: the vaults witnessed the deeds of mischief-makers and murderers, vagrants, and torturers. It’s one of Edinburgh’s most haunted sites, where restless spirits still tread.

First, you’ll follow your cloaked Mercat guide through the shadowy closes of Edinburgh’s Old Town. These are dark streets untouched by daylight, filled with a strange silence, their damp walls carrying only the faintest echo of the crowds on the high street you’ve left behind.

With the scene set, you’ll descend into the depths of the city – and listen in horror to its dark and sinister side. Torture. Murder. Hangings. From body snatchers to the real-life Jekyll and Hyde, you’ll be gripped by the true tales that lie hidden beneath Edinburgh’s streets.

Watch out for a cold breath on the back of the neck, a whisper, a flicker – then silence. It’s all the more chilling for being absolutely real: there are no tricks or jump scares on our ghost tours.

This tour is perfect for a peek into the city’s ghostly past because things don’t only go bump in the night. This tour may not be suitable for the faint of heart, or those who suffer from claustrophobia! If you choose not to go on the tour, the bus will pick you up at 11:30am on the way to SASA.

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The conference dinner will be held in First Coast restaurant: just a short walk from Haymarket – see map below. Named after a tiny village in Wester Ross, First Coast is a quality neighbourhood bistro offering fresh, tasty homemade food in a relaxed environment.

https://www.first-coast.co.uk

Any problems while in Edinburgh call

Greig: 0774 7788999 Gerry: 0782 5356028

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Ivette Acuña [email protected] Marie-Anne Barny [email protected] Amélie BEURY [email protected] Alison Blackwell [email protected] Doretta Boomsma [email protected] Greig Cahill [email protected] Jeremy Cigna [email protected] Guy Condemine [email protected] Triona Davey [email protected] Patrice de Werra [email protected] Yeshitila Degefu [email protected] Brice Dupuis [email protected] Jakub Fikowicz Krosko [email protected] [email protected] Vladimir Gorshkov [email protected] Mónica Gutierrez - Tjaart Hofman [email protected] Sonia Humphris [email protected] Miriam Kooman [email protected] Christophe Lacomme [email protected] Yves le Hingrat [email protected] Ewa Łojkowska [email protected] Rachel Mann [email protected] Minna Pirhonen [email protected] Marta Potrykus [email protected] Mirjam Prinz [email protected] Leighton Pritchard [email protected] Viviana Rivera-Varas [email protected] Åsa Rölin [email protected] Emily Ross [email protected] Gerry Saddler [email protected] Camila Sandoval - Gary Secor [email protected] Ian Toth [email protected] Jacquie van der Waals [email protected] Jan van der Wolf [email protected] Frederique VAN GIJSEGEM [email protected] Robert Vreeburg [email protected] Lauren Watts [email protected] Iris Yedidia [email protected]

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Euphresco III Dickeya/Pectobacterium Workshop

15 and 16 November 2018 NAK, Emmeloord, The Netherlands

Programme and Abstracts

Euphresco III Dickeya/Pectobacterium Workshop

15 and 16 November 2018 NAK, Emmeloord, The Netherlands

Local organisers  Jan van der Wolf, Wageningen UR  Miriam Kooman, NAK  Doretta Boomsma, HZPC B.V.

Coordinators Euphresco Dickeya/Pectobacterium project  Maria Bergsma-Vlami, NVWA  Jan van der Wolf, Wageningen UR

Coordinator Euphresco programme  Baldissero Giovani (EPPO)

Sponsors

Programme

Thursday 15 November (chair Miriam Kooman)

8.30 h Departure bus from Hotel Apollo, (Agoraweg 11, Lelystad) to the NAK (Randweg 14, Emmeloord)

9.00 h Welcome by Dr Eric Casteleijn (director NAK)

9.10 h Introduction workshop (Jan van der Wolf)

Surveys and ecology

9.25 h Leah Trsor. Characterization of Pectobacterium carotovorum subsp. brasiliense in Israel and seed treatments to control tuber soft rot

9.50 h Yeshitila Degefu. Extreme weather conditions and characterization of blackleg on potato during the 2018 cropping season in Finland

10.15 h Jianjun Hao. Tracking the bacteria associated with the outbreak of blackleg of potato in the Northeastern US

10.40 h Coffee/Tea break

11.15 h Marie Anne Barny. SRP in water environment, when, where and who is there?

11.40 h Robert Vreeburg. Virulence of different Pectobacterium brasiliense isolates tested at two different Dutch locations

12.15 h Vladimir Gorshkov. Non-conventional ways of pectobacteria adaptation to stress conditions

12.40 h Lunch

Genetic characterization of strains

13.30 h Nicole Hugovieux Cotte-Pattat. Abundance of Dickeya isolates in lakes from the French region La Dombes, including a new species, D. lacustris

13.55 h Minna Pirhonen. Pectobacterium polaris strains isolated in Finland

14.20 h Frédérique van Gijsegem. Dickeya diversity: Follow-up

15.05 h Coffee/Tea break

15.30 h Ewa Lojkowska. Genomic variability in the plant pathogenic bacterium Pectobacterium parmentieri deduced from de novo assembled complete genomes

15.55 h Jeremy Cigna. Contemporary pathogens: Description of Pectobacterium punjabense sp. nov. and sensitivity to biocontrol agents

16.20 h Malgorzata Waleron. Characterization of the two new species: Pectobacterium peruviense and P. zantedeschiae - can they pose a problem?

16.45 h Eef Jonkheer. Pangenome analysis and its application on Pectobacterium

17.10 h Valérie Hélias. Diversity and phylogeny of the Pectobacterium complex

17.35 h Jan van der Wolf. Metagenomics for detection and characterization of soft rot Pectobacteriaceae in air samples

18.30 h Departure for the workshop dinner (Urk)

Friday 16 November (chair Jan van der Wolf)

8.30 h Departure bus from Hotel Apollo, Lelystad to the NAK (Randweg 14, Emmeloord)

Plant pathogen interaction

9.00 h Iris Yedidia. Prospects on host specificity and differential pathogenicity of Pectobacterium spp.

Control

9.25 h Robert Czajkowski. Compatible mixtures of antagonistic bacteria developed specifically for biological control of Soft Rot Pectobacteriaceae (Pectobacterium spp. and Dickeya spp.) in potato (project PATBIOCON)

9.50 h Agata Motyka. Direct current atmospheric pressure glow discharge in possible agricultural applications

10.15 h Coffee/Tea

10.40 h Carina Eisfeldt. Survival of plant pathogenic bacteria in tile drainage water and the potential application of managed aquifer recharge in agriculture for safe reuse of irrigation water

11.05 h Ivette Acuna. Evaluation of the resistance to soft rot of commercial potato varieties in Chile

11.30 h Johannes Ransijn. Can complex systems approaches help to reduce disease incidence?

Diagnostics

11.55 Leighton Pritchard. Computational design of Pectobacterium diagnostics

12.20 Lunch

General discussion

- Trends in research on Pectobacterium/Dickeya

- Prospects on management of the disease

- Follow up Euphresco project and coordination of the follow up

- Next meeting

- Update “Erwinia book”

Tour de NAK

16.00 h Departure to Hotel Apollo

Saturday 17 November

10.00 – 13.00 h Sightseeing Amsterdam (see information at the end of the programme

Characterization of Pectobacterium carotovorum subsp. brasiliense in Israel and seed treatments to control tuber soft rot

Leah Tsror1, S. Manulis-Sasson2, S. Lebiush1, O. Erlich1, I. Galilov1, L. Chalupowicz2, M. Reuven2, O. Dror2, U. Zig3

1Department of Plant Pathology and weed Research, Agricultural Research Organization (ARO), 1Gilat Research Centre, 2The Volcani Center, 3Yaham Enterprises Ltd., Israel.

Potato cultivation in Israel is carried out in spring season using seed tubers imported from Europe and in fall-winter with local seeds, produced during the spring under the inspection of Plant Protection and Inspection Services. Pectinolytic bacteria, primarily Dickeya solani (Ds), Pectobacterium carotovorum (Pc), P. c. brasiliense (Pcbr) and P. parmentieri (Pp), are the causal agents of pre-emergence seed rot, blackleg and wilt and tuber soft rot causing economic losses in both seasons. Disease symptoms vary with climate and development and expression of the disease in field may be particularly favored under warm-climate conditions which prevail in Israel. Latent infections with Ds and Pcbr in imported seed material directly affect the crop yields in both seasons. Pcbr strains obtained from European imported seeds and from potato plants and progeny tubers grown in Israel were characterized by Pulse Field Electrophoresis Gel (PFGE) and by sequencing the gapA gene. A major group of 40 strains (out of 60 tested strains) produced the same pattern with either AvrII or I-CeuI. Their pathogenicity was evaluated by tuber maceration test. A considerable variation in maceration ability was observed within the tested strains related to the source of the plant material.

Seed tuber treatments were evaluated in reducing tuber rot during the fall-winter season in Israel, especially at early planting during September when temperatures are relatively high. The seed treatments included dry steam, oxolinic acid (Summit Agro International Ltd), mancozeb and MB5K (surface sterilizing agent, Adama Ltd) applied prior to planting. All treatments reduced seed decay or tuber rot, however, the results were inconsistent in field trials conducted in different years. The potential use of these treatments by farmers is considered.

Extreme weather conditions and characterization of blackleg on potato during the 2018 cropping season in Finland

Yeshitila Degefu

Natural Resources Institute Finland (LUKE)

Paavo Havaksen tie 3

FI-90014 University of Oulu, Finland

E-mail: [email protected]

According to the report of the Finnish Meteorological Institute, the summer from May to August 2018 in Finland was warmer than ever in the recorded history. July was the warmest month ever recorded in the history of the institute. There were a total of 27 hot days, days when temperatures rose over 25 ⁰C, in July alone. The summer was also characterized by exceptionally dry periods in most parts of the country. Extreme in both aspects. Therefore, the impact of these extreme weather conditions during the cropping season on the prevalence of blackleg on potato and the species of Dickeya and Pectobacterium predominant in the observed disease complex in the season is of particular interest. Diseases survey was conducted in the major seed potato growing areas in North Finland and ware potato production localities in the South Ostrobothnia near central Finland were carried out. Results from the characterization of observed outbreaks of blackleg from those areas and data obtained from routine lab tests for latent infection of seed tubers will be discussed.

Tracking the bacteria associated with the outbreak of blackleg of potato in the Northeastern US

Hao JJ1* Ge T1 Marangoni N1 Jiang H1 Johnson SB2 Larkin RP3

1School of Food and Agriculture,

2Cooperative Extension

3USDA-ARS, New England Plant, Soil, and Water Lab University of Maine, Orono, ME 04469, USA

*Email [email protected]

An outbreak of blackleg of potato, predominantly caused by Dickeya dianthicola, occurred in the Eastern states of the US beginning in 2015. Symptomatic potato plants were collected from Maine and the states where Maine seed tubers were planted and used to isolate blackleg pathogens. By analyzing 125 isolates of pectolytic bacteria with multilocus sequence analysis, Maine isolates were less diverse than those from the other states, indicating that outbreaks in other states may not have only originated from Maine seed tubers. Isolates collected from soil, non-potato plants, and surface water were analyzed but were not associated with the disease epidemic. It is probable that the outbreak resulted from multiple factors, which will be discussed.

SRP in water environment, when, where and who is there?

J Pédron1, Berge O2, ,Van Gisjegem F1, and Barny Ma1

1 Institute of Ecology and Environmental Science-Paris

University Pierre and Marie Curie - 4 place Jussieu - 75252 PARIS cedex 05

2INRA,Plant Pathology Research Unit, 84143 Montfavet, France

Soft Rot Pectobacteriaceae (SRP) diversity is well characterized on disease crop species in agricultural context. In contrast, their abundance, repartition and diversity outside the plant context remain largely unexplored. Early studies showed that SRP could be recovered from various non-host environments, such as water, soil or air. However, the taxonomic status of most of the isolated strains in these early works was not clearly assigned since most species were described later on. Therefore, is it still unclear which particular species could be recovered from various non-host environments. An effort to analyse the situation is particularly important with water since water could serve for irrigation purposes. We analysed the current situation at the scale of a whole catchment area, from pristine alpin water to irrigation water in arable land. Two years survey at the scale of the Durance catchment area was performed in 2016 and 2017. Sampling was performed four times a year in winter, spring, summer and fall on 21 sites on the main river and its tributaries. Plating water on CVP allowed us to survey the SPR concentration at each sites during the two consecutive years. Isolation of nearly 800 SPR and sequencing of the gapA housekeeping gene allowed us to characterize the isolated strains. The result of these two years survey will be presented.

Virulence of different P. c. subsp. brasiliense isolates tested at two different Dutch locations

1Robert Vreeburg, 1Eisse de Haan, 1Mathijs Nas1 (and others), 2Martzen ten Klooster, 2Doretta Boomsma, 3Jan van der Wolf, 4Johan van Vaerenbergh, 5Jack Gros

1NAK, Randweg 16, Emmeloord

2 HZPC B.V., Edisonweg 5, 8501 XG Joure

3 WUR, P.O. Box 16, 6700 AA Wageningen

4 ILVO, Burgemeester Van Gansberghelaan 92 bus 1, 9820 Merelbeke

5 Agrico Research, Burchtweg 17, Bant

Email: [email protected]

A Belgian-Dutch consortium of NAK, HZPC, WUR, ILVO and Agrico has tested different Pectobacterium carotovorum subsp. brasiliense (Pcb) isolates at two test sites in The Netherlands. The panel of Pcb isolates contained isolates from symptomatic potato plants, latently infected tubers, one isolate from insects and one from another latently infected crop. Tubers of the varieties Kondor and Spunta (class PB3) were vacuum inoculated with the different isolates both at the NAK and at HZPC, sharing the same potato tuber batch and isolates, but performing the experiment independently. Inoculated tubers were planted in four randomised blocks, with a total of 96 tubers per isolate. Wilting symptoms were scored weekly and accumulated symptom development was used as measure for virulence. Different isolates showed different virulence, with a similar picture at both sites. The virulence in Kondor was mirrored by the virulence in Spunta at the NAK site, but no additional symptoms above mock level were observed in Spunta at the HZPC site.

Virulence of a subset of isolates was tested by dipping inoculation, resulting in lower disease symptoms, but the isolates showed a similar ranking of the virulence as with vacuum inoculation. The virulence of the same subset was also tested on different varieties with minitubers, resulting in similar virulence results on the different potato varieties.

Some isolates that originate from symptomatic plants, and many isolates from latent tubers, were not or weakly virulent in the field experiments. One explanation for this is that the reduced-virulence of the isolates is an artefact of the isolation procedure. This hypothesis still needs to be tested in a new field experiment.

Non-conventional ways of pectobacteria adaptation to stress conditions

Vladimir Gorshkov1,2*, Olga Petrova1, Amina Daminova1, Marina Ageeva1, Natalia Gogoleva1,2, Bakhtiyar Islamov1, Vladimir Vorob`ev1,2, Yuri Gogolev1,2

1Kazan Institute of Biochemistry and Biophysics, Federal Research Center "Kazan Scientific Center of RAS"

2Kazan Federal University, Kazan, Russia

*Email [email protected], [email protected]

One of the major gaps in the understanding of soft rot pathogen biology is a limited information about the way these bacteria survive in various stressful ecological niches (e.g. soil, waste water, air, plant remnants) when being out of the host plant. The conventional concept states that in order to adapt, the non-spore-forming bacteria stop proliferation to divert resources to effectively resist stress factors by forming particular specialized cell morphotype or phenotype (e.g. viable but non-culturable cells, cell wall deficient forms, persisters, etc.). An alternative sigma factor RpoS as well as quorum sensing system (QS) are considered to be crucial molecular players in coordination of bacterial stress response. QS regulation of stress response defines that the phenomenon of bacterial adaptation is realized not at a cell but at the population level and requires cell-to-cell communication taking place only at high population density. Besides, it is believed that to effectively survive under stress effect, bacteria should be “pre-prepared” by moderate stress that stops fast proliferation and induces accumulation of storage compounds; in other words, bacteria of the stationary growth phase adapt much more effectively than those of log growth phase.

However, the bacteria may encounter stress factors either during active proliferation, or being at a low population density. Therefore, we proposed the existence of non- conventional ways of bacterial adaptation. We have revealed and described the phenomenon of adaptive proliferation, when stress response is associated not with the growth arrest but with a significant increase in population density even in the absence of an exogenous nutritional substrate. Adaptive proliferation occurs only in those cases when the population density is too low to implement cell-to-cell communication, and is evidently supported by endogenous cell resources. The increase of population density in this case proceeds until the cell titer reaches the quorum level and the bacteria acquire cross-protection. Additionally, we described the log phase cell adaptation compared to that of the stationary phase cells. Besides, although adaptation is considered to be a non-specific response, we have found that different stressors induce alternative regulatory cascades and that pectobacteria adaptation to some of them does not require functional rpoS gene. This study was partially supported by a grant МК-2191.2017.4 and Russian Science Foundation (project No. 17-14-01363).

Abundance of Dickeya isolates in lakes from the French region La Dombes, including a new species, D. lacustris

Hugouvieux-Cotte-Pattat N1*, Jacot-des-Combes C2, Briolay J2

1UMR5240 Microbiologie Adaptation and Pathogenesis,

2Plateforme DTAMB,

University Lyon 1, CNRS, INSA, F-69621Villeurbanne, France

*Email [email protected]

To better understand the natural diversity of Dickeya, a survey was performed in small lakes surrounded by wetlands in the French region of La Dombes. Several Dickeya isolates were obtained from lakes protected from direct agricultural inputs. Sequencing of the gene gapA revealed the presence of three already characterized species, D. zeae, D. chrysanthemi and D. aquatica. Other isolates belong to a phylogenetic group separated from the known Dickeya species. Genomic analysis supported the delineation of a new species for which the name Dickeya lacustris was proposed. The closest species is D. aquatica, previously isolated from rivers, suggesting that these two species have a common ancestor adapted to a water environment.

Pectobacterium polaris strains isolated in Finland

Miia Pasanen1, Thomas Schott2, Yeshitila Degefu3, Virpi Ahola4, Leighton Pritchard5 and Minna Pirhonen1

1Department of Agricultural Sciences, University of Helsinki, Helsinki, Finland

2Herne Genomic, Neustadt, Germany

3Luke Natural Resources Institute Finland, Oulu, Finland

4Department of Biosciences, University of Helsinki, Helsinki, Finland

5Information and Computational Sciences, The James Hutton Institute, Dundee, Scotland, United Kingdom

Pectobacterium polaris is a newly identified pectinolytic and highly virulent plant pathogen isolated in Norway from potato samples. Five Pectobacterium isolates identified from potato stems in Finland were previously characterised as Pectobacterium carotovorum subsp. carotovorum but were not recognized in PCR tests that detect various Pectobacterium species. Average Nucleotide Identity (ANI) revealed that the representatives of the Finnish isolates, s0416 and s0421, shared 96% identity with Pectobacterium polaris strains NIBIO 1006T and NIBIO 1392, and over 99% identity with P. polaris strain NCPPB 3395 and isolate YC T1 still included into P. carotovorum in NCBI. However, based on MLSA and GGDC analyses and genome comparisons, the Finnish isolates and NCPPB 3395 were found to be differentiable from the P. polaris isolates NIBIO 1006T and NIBIO 1392, possibly representing a separate subspecies. The implication of these results for the taxonomy of the Finnish isolates is discussed.

Dickeya diversity: Follow-up

Frédérique Van Gijsegem iEES Paris (Institute of Ecology and Environmental Sciences), INRA-Sorbonne Universités UPMC Paris 06, UMR 1392, F-75005, Paris, France

*Email [email protected]

Eight species have been described in the Dickeya genus sofar: D. dadantii, D. dianthicola, D. zeae, D. chrysanthemi, D. paradisiaca, D. solani, D. aquatica and D. fanghzondai. This was however based mostly on strains isolated from diseased plants. To analyse the Dickeya diversity in another environment, we performed an extensive survey of the population of pectinolytic bacteria collected in a water canal near Avignon in the South of France. Most Dickeya species were found as well as some isolates belonging to a yet unidentified genomic clade including a strain isolate from a lake in Asia. We also isolated different D. solani strains. D. solani was mainly isolated from potato and ornamentals and appeared to be clonal, differing only by a few dozen SNPs/InDels except for one divergent strain. A recent reclassification of all former Erwinia chrysanthemi strains present in the CFBP collection into the different Dickeya species allowed the identification of a new D. solani strain that was isolated from a previously non-identified host – tomato - in Guadeloupe. A similar analysis of the Swiss collection of former E. chrysanthemi strains isolated from potato since the 1980s and our water survey led to the isolation of other D. solani strains presenting different levels of diversity. This intra-specific diversity will be discussed.

Genomic variability in the plant pathogenic bacterium Pectobacterium parmentieri deduced from de novo assembled complete genomes

Ewa Lojkowska1, Sabina Zoledowska1, Agata Motyka1, Wojciech Sledz1, Alessio Mengoni2

1Department of Biotechnology, Intercollegiate Faculty of Biotechnology UG and MUG, Poland.

2Department of Biology, University of Florence, Sesto Fiorentino, Florence, Italy

Pectobacterium parmentieri is a causative agent of diseases in economically important crops (e.g. potato) in a wide range of different environmental conditions, encountered in Europe, North America, Africa, and New Zealand. Severe disease symptoms result from the activity of P. parmentieri virulence factors, such as plant cell wall degrading enzymes. Interestingly, we observe significant phenotypic differences among P. parmentieri isolates regarding the abilities to macerate plant tissue and activities of virulence factors. The pan-genome study was performed on 15 genomes:12 de novo assembled and three reference strains: P. parmentieri CFBP 8475T, P. parmentieri SCC3193, P. parmentieri WPP163. The pan-genome includes 3 706 core genes and 3 315 dispensable genes; within them 1 468 accessory genes, and 1 847 unique genes. The presence of well- known genes encoding virulence factors was observed in the core genome fraction, but some of them were located in the dispensable genome. A significant fraction of horizontally transferred genes, virulence-related gene duplications, as well as different CRISPR arrays were found, which can explain the observed phenotypic differences. We can hypothesize that a large number of the genes in the dispensable genome and significant genomic variation among P. parmentieri strains could be the basis of its widespread diffusion and adaptation to different environments.

Contemporary pathogens: Description of Pectobacterium punjabense sp. nov. and sensitivity to biocontrol agents

Cigna Jérémy*1-2, Robic Kévin1-2, Dewaegeneire Pauline1, Laurent Angélique1, Gobert Virginie1, Le Hingrat Yves1, Beury-Cirou Amélie1, Hélias Valérie1, and Faure Denis2.

1 National Federation of Seed Potato Growers (FN3PT-RD3PT), 75008 Paris, France

2 CNRS-I2BC, ANR-15-CE21-0003, 1 avenue de la terrasse, 91198 Gif-sur-Yvette, France

*Email: [email protected]

The characterization of isolates from the Pectobacterium genus, originating from blackleg symptoms in Pakistani potato fields have conducted to the recent description of a new species called P. punjabense in reference to the area where it was isolated. Here we present the MLSA studies, DNA-DNA hybridization and ANI values that clearly revealed the isolate SS95T as a member of the new P.punjabense species. In complement, an in- silico analysis allowed us to identify specific genomic region present in P. punjabense and absent in the nearest P. parmentieri and P. wasabiae species. Phenotypic assay using various carbon sources indicates the same features for P. punjabense and P. parmentieri type strains. Maceration tuber assay with P. punjabense SS95T was compared to others Pakistani isolates. Results revealed a level of aggressiveness equivalent to the P. parmentieri isolate and lower than the D. dianthicola and P. atrosepticum isolates. Finally, P. punjabense strains were occasionally identified in our collection and elsewhere in Europe, showing that this species is not restricted to Pakistan. Results will be discussed.

At the same time, biocontrol experiments were conducted in order to control the blackleg disease. One of the objectives is to study the sensitivity of contemporary pathogens regarding three biocontrol agents previously selected. In this context, 600 pectinolytic bacteria were isolated since 2013 from blackleg symptoms in French potato fields. After identification of all the isolates with “gapA phylogeny”, a sample group of 41 isolates was selected, including one new P. punjabense isolate. Finally, their sensitivity regarding the biocontrol agents was studied. Results of antibiosis test showed a greater inhibition zone with one of the biocontrol agents compared to the others, whatever the pathogen isolate tested.

Characterization of the two new species: P. peruviense and P. zantedeschiae - can they pose a problem?

1 1 1 2 2 3 4 Waleron M , Misztak A Waleron MM , Franczuk M , Jonca J , Wielgomas B , Mikiciński A , Popović T5 and Waleron K2

1 Department of Biotechnology, University of Gdansk, Poland

2 Department of Pharmaceutical Microbiology, Medical University of Gdansk, Poland

3 Department of Toxicology, Medical University of Gdansk, Poland

4 Research Institute of Horticulture, Skierniewice, Poland

5 Institute for Plant Protection and Environment, Belgrade, Serbia;

* e-mail: [email protected]

The genus Pectobacterium was proposed for the first time by Waldee in 1945. Since then, the taxonomy of this genus underwent extensive remodelling. The appearance of the new and more affordable sequencing technologies resulted in the progressive increase in data pool allowing for better insight into variety within this genus. Studies based on the genomic data revealed the need for reclassification of several previously misassigned strains. As a result of the new species have been described, among them Pectobacterium peruviense and Pectobacterium zantedeschiae.

The new species P. peruviense gather strains isolated from potatoes tubers cultivated in the high altitude in Peru that can infect and efficiently macerate tissue in temp. range 12 – 28 °C. What’s more, the bacteria belonging to this species are not only resistant to low temperature, but also to high salinity. Recently new P. peruviense strains were isolated from alpine rivers in France. The strains of P. zantedeschiae, which were isolated from Calla lily, are highly virulent and can infect both monocotyledonous and dicotyledonous plants. Strains from this species tolerate a wide range of salinity, pH, and water availability and also they show resistance to some antibiotics, isothiocyanate and bile salts.

Financed: NCN2015/17/B/NZ9/01730

Pangenome analysis and its application on Pectobacterium

Eef Jonkheer1, Sandra Smit1, Dick de Ridder1, Balázs Brankovics2, Ilse Houwers2, Jan van der Wolf2, Peter Bonants2, Robert Vreeburg3,Theo van der Lee2

1. Bioinformatics group, Wageningen University, Droevendaalsesteeg 1, 6708PB Wageningen, The Netherlands

2. Biointeractions & Plant Health, Wageningen Plant Research, Droevendaalsesteeg 1, 6708PB Wageningen, The Netherlands

3. Nederlandse Algemene Keuringsdienst, Randweg 14, 8304 AS Emmeloord

E-mail: [email protected]

Next-Generation Sequencing (NGS) results in many, sometimes hundreds or thousands, of high-quality genome assemblies for a single species. Current reference based genome analysis pipelines cannot deal with such numbers effectively. To manage, store and exploit these data we need new strategies. Previous studies have shown that in a single species we can identify the core genome (present in all), the accessory part (present in some) and unique parts, therefore a single reference genome is simply inadequate to capture the genotypic diversity. To analyse multiple genomes simultaneously we developed PanTools (Sheikhizadeh et al,2016 ), an innovative platform for pangenomic analysis that is scalable and computational efficient. PanTools uses a localized de Bruijn graph as a pangenome representation and stores the data in a Neo4j graph database. This graph not only stores the sequence data, but by including annotations we can easily traverse the graph and interrogate the data to address biological questions. Within PanTools fundamental but complicated questions such as the identification of orthologous genes can be performed in a fraction of the time compared to popular methods such as Orthofinder. In addition PanTools allows linking metadata associated with the genomes to identify communalities between genomes that share a phenotype or other feature.

We will present some first results of our approach applying PanTools on the analysis of bacterial genome content and organization. We constructed a pangenome of 40 Pectobacterium brasiliensis of both in-house sequencing data as genomes from NCBI. Within this pangenome we performed various analyses such as calculation of ANI scores, clustering of proteins and estimation of the core, accessory and unique genome. In the 40 genomes we can distinguished at least three different clusters. We found 200 orthologous groups of genes specific to one of these clusters. The genes in these groups typically are physically linked in the genome. Using functional annotation tools we were able to identity candidates genes that could be important for the infection.

This project is financed by the PPS innovatieve diagnostiek. With partners NAK Emeloord, Naktuinbouw and BKD

Diversity and phylogeny of the Pectobacterium complex

Khaoula Chawki1,2, Angélique Laurent1,2, Géraldine Taghouti3 , Emma Caullireau, Marion Fischer-le Saux3, Yves Le Hingrat1, Didier Andrivon2 , Perrine Portier3, Valérie Hélias1,2

1 FN3PT/RD3PT, French Federation of Seed Potato Growers, 43-45 rue de Naples, F- 75008 Paris, France

2 INRA, UMR1349 IGEPP, F-35653 Le Rheu Cedex, France

3 CIRM-CFBP, IRHS, INRA, AGROCAMPUS-Ouest, Université d’Angers, SFR 4207 QUASAV, 42 rue Georges Morel, 49071 Beaucouzé cedex, France

Keywords : taxonomy, soft rot, MLSA, blackleg, certification

Pectobacterium contains to this day 11 official species after recent new assignments. Field surveys conducted annually in France since 2003, coupled with diversity studies of the resulting strain collections, showed that blackleg symptoms are associated with a much larger range of species than the one (P. atrosepticum) historically almost exclusively held responsible for these symptoms in Europe. We thus aimed at analyzing more deeply the diversity and the phylogeny of Pectobacterium, particularly strains isolated from field symptoms on potato, and at evaluating changes within the P. carotovorum (P.c.) group during the last decades. Refining the taxonomy of the Pectobacterium groups was needed to develop specific detection tools, useful for testing and certifying seed lots. A MLSA was performed using recA, dnaX and leuS genes on 384 strains from the FN3PT and the CIRM-CFBP collections; 53 Pectobacterium sequences from databases were used as supplementary data. The already described species including the recently described P. polaris and the subspecies P. c. odoriferum, P. c. brasiliense, P. c. actinidiae were clearly defined by the MLSA results. In contrast, strains from P. carotovorum subsp. carotovorum showed a high genetic diversity in accordance with our earlier sequencing work on PelY. The results showed that P. carotovorum subsp. carotovorum strains were distributed in several groups, all not being previously described. Among them, one corresponded to the “putative P. maceratum”. Additionally, some orphan strains were identified, that were not allocated to any specific cluster, except one, belonging to the P. punjabense species (see Cigna et al.).

Metagenomics for detection and characterization of soft rot Pectobacteriaceae in air samples

Van der Wolf, J.M.1*, Dullemans, A.1, Krijger, M.1, Putra, G.1, Warris, S1. & Fokkema, J.2

1Wageningen University & Research, NL

2NAO (NAO; Dutch Organisation of Potato Merchants), NL

*Email [email protected]

In the past, it has been shown that soft rot Pectobacteriaceae can be spread from infected potato fields via aerosols, generated due to splashing water or during (mechanical) haulm destruction. Analysis of sampled aerosols can be important in studies on transmission of the pathogen but may also be efficient in the diagnosis of fields. In our studies, 24 air samples were collected with a Coriolis air sampler in different seed potato crops after which samples were incubated for 72 h in Pectate Enrichment Broth. During mechanical haulm destruction, samples were found positive with TaqMan assays specific for Pectobacterium brasiliense (%) and P. parmentieri (%). Air samples collected during plant growth before haulm destruction were all negative in the TaqMan assays, also samples collected during selection and after spraying chemicals. The application of metagenomics was subsequently explored on 12 of these air samples. In metagenomics, samples are analysed in a unbiased way and allow to discriminate pathogens at the strain level. Total DNA was extracted from the enriched air samples and sequenced (HiSeq). Sequences were mapped against reference genomes of soft rot Pectobacteriaceae. The presence of P. brasiliense and P. parmentieri was confirmed, but also P. polaris, P. carotovorum and likely P. odoriferum were detected for which no species specific TaqMan assays are developed yet. We further found strong indications that in samples different variants of P. brasiliense can be present. The use of metagenomics in diagnostics, in particular in forensic studies will be discussed.

This project was financed by the Dutch Ministry of Economic affairs via the TopSector Programme and the Dutch seed potato industry via the NAO

Prospects on host specificity and differential pathogenicity of Pectobacterium spp.

Nirmal Khadka1,2, and Iris Yedidia2*

1Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel;

2 Institute of Plant Sciences, Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel; *email: [email protected]

With the “reputation” of a wide-host-range pathogen, specificity or “affinity” to a certain host is relatively a less studies aspect of Pectobacterium infection. Most microbial ecology studies of the genus were based on crop plants (vegetables or ornamentals) with potato being the most funded and studied system. In the last decades, with an ever improving genetic tools for phylogenetic classification, an increasing number of new species and subsp. in the genus were discovered. Despite all these tools, little attention has been devoted to the performance of different species on their respective hosts, their interaction with the host environment on a biochemical level, and the differential host responses towards different strains of Pectobacterium. Here, we characterized a differential response of isolates from potato (P. carotovorum, and P. carotovorum subsp. brasiliense) or calla lily (P. aroidearum) on two hosts: Brassica oleracea (cabbage) and Zantedeschia aethiopica (calla lily). The results revealed clear differential expression of virulence determinants within the bacterial strains in response to host’s extracts; these were related to each strain’s competence on its respective host. A similar trend of differential gene expression was demonstrated in the induced- resistance response of the plant hosts to the different bacterial strains.

Compatible mixtures of antagonistic bacteria developed specifically for biological control of Soft Rot Pectobacteriaceae (Pectobacterium spp. and Dickeya spp.) in potato (project PATBIOCON)

Dorota M. Krzyzanowska 1§, Tomasz Maciag1 §, Joanna Siwinska 2, Marta Krychowiak 3, Sylwia Jafra 1 and Robert Czajkowski 3 *

1Laboratory of Biological Plant Protection,

2 Laboratory of Plant Protection and Biotechnology,

3 Laboratory of Biologically Active Compounds,

Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Gdansk, Poland

§ - these authors contributed equally to this work

*Email: [email protected]

Potato (Solanum tuberosum L.) is the fourth main food crop in the world after rice, maize and wheat. The very intensive global potato cultivation together with the international potato tuber market results in the risk of transmission and spread of various diseases. Diseases may influence potato production in any stage of crop growth and storage. Likewise, the diseases may affect tubers, above-ground plant parts (foliage) or both. From all diseases affecting potato, the ones caused by pectinolytic Soft Rot Pectobacteriaceae (SRP: Pectobacterium spp. and Dickeya spp.): blackleg of stems during plant cultivation and soft rot of tubers in storage are among the most important bacterial potato diseases recognized in potato production. These diseases led to estimated losses of ca. 10 to 40% crop (even up to ca. 250 million Euro) annually worldwide. At the moment there are no commercial control products to be used against pectinolytic Pectobacterium and Dickeya in agriculture. This is due to the fact that during infection the bacteria are readily present in protective niches (inside plant vascular tissues) and the majority of chemicals and physical control measures work only superficially and cannot penetrate to the tissues located deeper inside plants. The PATBIOCON project aims to use biocontrol measures to protect agricultural and ornamental plants from infections with pectinolytic bacteria. For this, we have selected beneficial bacteria occupying the same niche and antagonistic to Pectobacterium and Dickeya and evaluated them individually and in compatible mixtures against the pectinolytic bacteria under disease-provoking conditions (high temp., high humidity, high pathogen load, hypoxia). Application of a combination of (compatible) beneficial bacteria with different modes of antagonistic action provided significant protection of plant tissues against Pectobacterium and Dickeya (reduction of tuber soft rot by 46% (p=0.0016)). The selected antagonists were additionally characterized

22 for features important for their viable commercial applications including growth at different temperatures, resistance to antibiotics and potential toxicity towards Caenorhabditis elegans. The implications for control of soft rot caused by SRP with the use of the composition (mixture) of antagonists will be discussed.

The mixture of bacterial isolates described herein, for protection of potato tubers and ornamental plants against soft rot caused by pectinolytic Pectobacterium spp. and Dickeya spp., is the object of the patent application P.423806, which has been filed with the Polish Patent Office by University of Gdansk, Poland with inventors Robert Czajkowski, Dorota M. Krzyzanowska, Tomasz Maciag, Joanna Siwinska and Sylwia Jafra

The work was financially supported by the National Centre for Research and Development, Poland (Narodowe Centrum Badań i Rozwoju, Polska) via a research grant LIDER VI (LIDER/450/L-6/14/NCBR/2015) to Robert Czajkowski.

Direct current atmospheric pressure glow discharge in possible agricultural applications

Motyka-Pomagruk A1* Dzimitrowicz A2 Jamroz P2 Babinska W1 Pohl P2 Lojkowska E1 and Sledz W1

1Department of Biotechnology, University of Gdansk and Medical University of Gdańsk, Gdansk, Poland

2 Department of Analytical Chemistry and Chemical Metallurgy, Wroclaw University of Science and Technology, Wroclaw, Poland

*Email [email protected]

Diseases caused by phytopathogenic bacteria are responsible for significant economic losses. Therefore high interest is attributed to development of novel eradication methods, not only to be applied in the fields but also in greenhouses, storage areas or having in mind decontamination of waterways or post-industrial wastes. For the latter purpose we herein propose a direct current atmospheric pressure glow discharge (dc-APGD)-based liquid sterilisation system (Polish patent application no. P.419246), utilization of which by generation of UVA, UVB and UVC in addition to the production of ROS and RNS (NOx, NH,

H2O2, O2, O and OH) led to complete eradication of Clavibacter sepedonicus IFB9034, Dickeya solani IFB0099, and Xanthomonas campestris pv. campestris IFB 9022 from suspensions of OD600 ≈ 0.1 and at least 3.43 logarithmic reduction in the population densities of Pectobacterium atrosepticum IFB5103 and Pectobacterium carotovorum subsp. carotovorum IFB5118. Besides, dc-APGD was also applied for the synthesis of silver nanostructures (AgNPs), stabilized either by pectins (PEC), fructose (FRU) or sodium dodecyl sulphate (SDS). The obtained PEC-AgNPs and SDS-AgNPs exhibited antibacterial properties against Dickeya and Pectobacterium spp strains with minimal inhibitory concentrations (MIC) of 5.5 mg∙L−1 and 0.75–3 mg∙L−1, respectively. On the other hand, FRU-AgNPs were used against Erwinia amylovora IFB9037, Clavibacter michiganensis IFB9038, Ralstonia solanacearum IFB8019, X. c. pv. campestris IFB9022 and D. solani IFB0099 strains, showing MICs of 1.64-13.1 mg L-1 depending on the species tested. We postulate that dc-APGD might find numerous applications in the agricultural sector contributing to improvement of plant disease control.

Plant pathogen removal from tile drainage water by managed aquifer recharge for safe reuse as irrigation water in salinized agricultural areas

Eisfeldt, Carinaa, Van der Wolf, Jan M.b, van Breukelen, Boris. M.a, Schijven, Jack F.c, Medema, Gertjana a TU Delft, Faculty of Civil Engineering and Geosciences, Building 23, Stevinweg 1, 2628 CN Delft, The Netherlands b Wageningen Plant Research, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands c Utrecht University, Faculty of Geosciences, Heidelberglaan 2, 3584 CS Utrecht, The Netherlands

The presence of bacterial plant pathogens in waterways and their dissemination through irrigation water pose a threat to crop production. A natural solution to provide safe irrigation water is managed aquifer recharge (MAR) for agriculture. Here, tile drainage water (TDW) is collected and infiltrated in brackish aquifers, resulting in a fresh water ‘bubble’ in the subsurface which gives farmers access to sufficient fresh water to irrigate their crops even in times of drought. The TDW may contain plant pathogens which could be present in the recycled water. To prevent the occurrence of crop diseases, the removal of selected plant pathogens in TDW and during aquifer soil passage will be analysed. We focused on three plant pathogenic bacteria, namely Ralstonia solanacearum, Dickeya solani and Pectobacterium carotovorum sp. carotovorum that are all present in the Netherlands but also depict a worldwide problem as they cause high crop losses associated with immense economical costs. Their die-off in natural TDW was studied under representative aquifer conditions in microcosm experiments and at a starting concentration of 104 CFU/mL. First results under aerobic conditions show that all bacteria were not detected anymore in 0.1-ml samples within 14 days at 10 ⁰C using viable cell counting, corresponding to 3 log10 reduction by die-off. D. solani and P. carotovorum were no longer detected within 6 days at 25 ⁰C, but R. solanacearum was more persistent at 25 ⁰C and was detectable in 0.1 ml up to 25 days. Together with results from soil column experiments and field tests, these data will be used to predict the fate of pathogens during MAR using quantitative microbial risk assessment. Guidelines will be developed to demonstrate the feasibility of MAR in agricultural settings, its potential to deliver safe irrigation water and thereby fighting water scarcity and preventing food losses.

Since July 2017 Carina Eisfeld is pursuing her PhD research within the project of AGRIMAR which stands for agricultural managed aquifer recharge. Her part in the project is to analyse the water quality regarding plant pathogens to prevent the spread of plant diseases and consequently the loss of food crops. The goal is to proof that MAR technology is a safe and sustainable process that fights water scarcity in saline delta regions and provides safe water for irrigation.

Evaluation of the resistance to soft rot of commercial potato varieties in Chile

Acuña I., Sandoval C., Mancilla S. and A. Bermúdez.

Institute of Agricultural Research INIA Chile

E-mail: [email protected]

Blackleg and soft rot, caused by Pectobacterium atrosepticum and P. carotovorum carotovorum, is a very important disease in potato production in Chile. This disease is responsible of 5 to 20% of yield losses, according to seed quality, environmental conditions and variety susceptibility. The objective of this study was to determine the variety susceptibility to soft rot on potato tubers. Seventeen potato varieties were evaluated using half tuber inoculation method with 1X 108 cfu of P. atrosepticum or P. c. carotovorum. Inoculated tubers were incubated at 27°C for 96 hrs. Disease severity was estimated measuring the rotten tissue volume per tuber. Atlantic, Rayun, Cornado and Patagonia INIA were the most susceptible, while Infinity, Romano and Red Sonia were the most resistant.

Soft rot in potato crops. Can complex systems approaches help to reduce disease incidence?

Johannes Ransijn

Dutch General Inspection Service for Agricultural Seed and Seed Potatoes (NAK)

Email: [email protected]

Infection of potato plants with soft rot causing pathogens (SRP’s, Pectobacterium and Dickeya species), is generally assumed to come from infected seed tubers (vertical transmission). There are several hints that horizontal transmission between potato lots or between potato and non-potato hosts plays a bigger role in the spread of SRP’s than previously thought. The presence of multiple SRP species and species complexes and the recent spread of a previously absent species are indicative for the complexity and dynamism of the disease system.

The potential power of epidemiological and complex systems approaches has hardly been used for investigation of the potato soft-rot disease system. We are only beginning to apply methodologies developed for plant, human and veterinary epidemiology on the extensive data from routine phytosanitary surveys from the Dutch seed potato certification programme. An outline for future research and preliminary results will be presented and discussed.

The main objectives of the epidemiological research on SRP’s are:

1) To assess the relative contribution of different infection pathways and the survival of SRP’s on infected potato lots

2) To infer the importance of risk factors contributing to the spread, survival and virulence of different SRP’s

3) To guide field and laboratory experiments and generate data-based hypotheses on the epidemiology and ecology of SRP’s.

4) To guide policies to reduce the incidence of softrot and blackleg in potato crops by, for example, agronomic measures, field selection or risk-dependent intensity of inspection

Computational design of Pectobacterium diagnostics

Pritchard L*, Humphris S and Toth I

James Hutton Institute, Invergowrie, Scotland, DD2 5DA

*Email [email protected]

We report on progress in 2018 in diagnostic qPCR amplicon and metabarcoding marker design for Pectobacterium, using the PDP software package. We have designed over 40 novel diagnostic primer sets, intended to be specific to species: P. atrosepticum, P. brasiliense, P. wasabiae, P. parmentieri, P. oderiferum, P. polaris, P. betavasculorum, and universal to the genus Pectobacteriaceae. We have determined new candidate primers that were absolutely specific to P. atrosepticum (2 primer sets), P. brasiliense (1 set), P. betavasculorum (4 sets), and P. oderiferum (2 sets) by validation against a panel of 171 bacterial isolates. We will describe new features and capabilities of the PDP design software, including the ability to design candidate metabarcoding markers for within- species diversity estimation.

Additional information

Transport

There is a direct train connection from Schiphol to the railway station in Lelystad. The travel takes about 50 minutes. The Apollo hotel in Lelystad (Agoraweg 11) is located at a walking distance from the railway station (6 minutes).

The train tickets can be bought at the ticket office at Schiphol airport or the ticket machine. Information is provided on the following website: https://www.rijdendetreinen.nl/en/information/buying-train-tickets-netherlands, In case you want to buy a ticket in advance, use the following link: https://www.ns.nl/en

On Thursday and Friday, the transport by bus from the hotel in Lelystad to the NAK and back is arranged. In the morning the bus will pick you up at the hotel at 8.30 h. The costs of the bus transport is generously paid by HZPC.

In case you miss the bus or come late. NAK is located at Randweg 14, 8304 AS Emmeloord, Nederland, T T +31 527 635 400. The mobile telephone number of Miriam Kooman is +31.651191133 and of Jan van der Wolf +31.6.10028194.

Lunch and workshop dinner

The lunch on Thursday and Friday and the workshop dinner at Thursday night are kindly offered by the NAK.

Saturday, sight-seeing Amsterdam. a bike tour: https://a-bike.eu/product/amsterdam-sightseeing-bike- tour/?gclid=EAIaIQobChMImK_2r_-U3QIVCed3Ch1ASQLaEAAYAyAAEgKRpvD_BwE b. a boat tour: http://classiccanalcruises.com/ (for those that are hesitating to bike in November in the Netherlands)

The costs will be between 20 and 30 euro and are at your own expense.

Both tours will end approximately 13.00 h. If people like to stay for the lunch, I’m sure that they find their way in the many interesting cafés and restaurants.

List of Participants

First name Surname Affiliation E-mail

1 Ivette Acuna INIA [email protected]

2 Marie-Anne Barny Sorbonne Université [email protected]

3 Doretta Boomsma HZPC [email protected]

4 Greig Cahill SASA [email protected]

5 Jérémy Cigna FN3PT/RD3PT [email protected]

6 Nicole Cotte-Pattat Insa Lyon [email protected]

7 Robert Czajkowsi University of Gdańsk [email protected]

8 Yeshitila Degefu Natural Resources Inst. [email protected] (man) Finland

9 Carina Eisfeld TU Delft [email protected]

10 Joost Gierkink HZPC [email protected]

11 Vladimir Gorshkov Kazan Scientific Center [email protected]

12 Jianjun Hao Maine [email protected]

13 Valérie Hélias FN3PT/RD3PT/INRA [email protected]

14 Sonia Humphris James Hutton Institute [email protected]

15 Eef Jonkheer WUR [email protected]

16 Miriam Kooman NAK [email protected]

17 Viola Kurm WUR [email protected]

18 Ewa Lojkowska University of Gdańsk [email protected]

19 Agata Motyka-Pomagruk University of Gdansk [email protected]

20 Minna Pirhonen Uhelsinki [email protected]

21 Leighton Pritchard James Hutton Institute [email protected]

22 Johannes Ransijn NAJ [email protected]

23 Åsa Rölin Potatis Konsult [email protected]

24 Martzen ten Klooster HZPC [email protected]

25 Wojciech Śledź University of Gdansk [email protected]

26 Leah Tsror Volcani Agri [email protected]

27 Frederique van Gijsegem Sorbonne Université [email protected]

28 Theo van der Lee WUR [email protected]

29 Jan van der Wolf WUR [email protected]

30 Robert Vreeburg NAK [email protected]

31 Malgorzata Waleron University of Gdansk [email protected]

32 Iris Yedidia Volcani Agri [email protected]